Novel compositions and methods of screening for B cell activity modulators

ABSTRACT

The invention provides for the identification of all genes, whether known or novel, which are differentially expressed within and among B cells, making possible the characterization of their temporal regulation and function in the B cell response and/or in B cell mediated disorders. Expression profiles, nucleic acids and proteins are provided for differing states of B cells, including resting, naive, activated, tolerant and immunosuppressed B cells. The present invention makes possible the identification and characterization of targets useful in prognosis, diagnosis, monitoring, rational drug design, and/or therapeutic intervention of immune system disorders.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT TECHNICAL FIELD

[0001] The invention relates to the identification of expressionprofiles and the nucleic acids involved in B cell activation,immunosuppression and immunological tolerance and to the use of suchexpression profiles and nucleic acids in methods for identifyingcandidate agents which modulate B cell activity.

BACKGROUND OF THE INVENTION

[0002] B lymphocytes (also referred to as B cells) mature within thebone marrow and leave the marrow expressing a unique antigen-bindingmembrane receptor. The B-cell receptor is a membrane-boundimmunoglobulin glycoprotein. When a B cell encounters the antigen forwhich its membrane-bound antibody is specific, the cell begins to dividevery rapidly; its progeny differentiate into memory B cells and effectorcells called plasma cells. Memory B cells have a longer lifespan andcontinue to express membrane-bound antibody with the same specificity asthe original parent cell. Plasma cells do not produce membrane-boundantibody but instead produce the antibody in a form that can besecreted.

[0003] Immunologic tolerance is a specific state of non-responsivenessto an antigen. Immunologic tolerance generally involves more than theabsence of an immune response; this state is an adaptive response of theimmune system, one meeting the criteria of antigen specificity andmemory that are the hallmarks of any immune response. Tolerance developsmore easily in fetal and neonatal animals than in adults, suggestingthat immature T and B cells are more susceptible to the induction oftolerance. Moreover, studies have suggested that T cells and B cellsdiffer in their susceptibility to tolerance induction. Induction oftolerance, generally, can be by clonal deletion or clonal anergy. Inclonal deletion, immature lymphocytes are eliminated during maturation.In clonal anergy, mature lymphocytes present in the peripheral lymphoidorgans become functionally inactivated.

[0004] Treatment of transplant and autoimmune patients often includessuppression of lymphocyte activation by tacrolimus (FKS06) orcyclosporin, both inhibitors of calcineurin. Borel, et al., (1976)Agents Actions 6: 468-75; Kino et al., (1987) J. Antibiot. (Tokyo) 40,1256-65; Ho, et al, (1996) Clin. Immunol. Immunopathol. 80, S40-5; andRuhlmann and Nordheim, (1997) Immunobiology 198, 192-206. Whileeffective during therapy, these compounds do not allow(re-)establishment of immunological tolerance to the offendingautoantigen and instead can inhibit tolerance induction. Prud'homme andVanier, (1993) Clin. Immunol. Immunopathol 66:185-92. Accordingly, thedevelopment of drugs that could induce tolerance would be desirable.

[0005] Therefore, it is an object of this invention to identify theexpression profiles which are unique to B-cell tolerance, activation andimmunosuppression. It is further an object to use the expressionprofiles in assays to identify agents which can be used in themodulation of B cell activity including B cell tolerance, activation,immunosuppression, mitosis, apoptosis, differentiation and migration. Itis further an object to use the expression profiles as diagnostics toidentify B cells which are abnormal. It is further an object to provideassays to identify agents for the treatment of B cell related disorders.

SUMMARY OF THE INVENTION

[0006] In one aspect of the invention, the identification of all genes,whether known or novel, which are differentially expressed within andamong B cells are provided, making possible the characterization oftheir temporal regulation and function in the B cell response and/or inB cell mediated disorders. Thus, expression profiles, nucleic acids andproteins are provided for differing states of B cells, includingresting, naive, activated, tolerant and immunosuppressed B cells. Thus,the present invention makes possible the identification andcharacterization of targets useful in prognosis, diagnosis, monitoring,rational drug design, and/or therapeutic intervention of immune systemdisorders.

[0007] The invention provides methods of screening drug candidates. Suchmethods entail providing a cell that expresses an expression profilegene selected from the group Egr-1, Egr-2, Nur77, c-myc, MIP-1a,MIP-1b,BL34, gfi-1, NAB2, neurogranin, SLAP, A1, E2-20K, SATBl, Cctq,kappa V, pcp-4, TGIF, CD83, ApoE, Aeg-2, CD72, cyclin D2, 1ck, MEF-2C,bmk, IgD, Evi-2, vimentin, CD36, c-fes, c-fos, TRAP, hIP30, Ly6E.1,LRG-21, Fos B, gadd153, maik, Ah-R, C/EBP beta, EZF, TIS7, TIS11,TIS11b, LSIRF, MKP1, PAC-1, PEP, MacMARCKS, SNK, Stra13, kir/gem, EB12,IL1-R2, MyD116, RP 105, UPAR, 4F2, hRab30, Id3, BKLF, LKLF, EFP, bcl-3,caspase 2, GILZ, hIFI-204, hRhoH, TRAF5, LT-beta, IFNg-RII, gadd45,CDC47, NAG, scd2, kappa 0 ig, iap38, G7e, B29, and SCD2. A drugcandidate is added to the cell. The effect of the drug candidate on theexpression of the expression profile gene is then determined.

[0008] In some methods the level of expression in the absence of thedrug candidate to the level of expression in the presence of the drugcandidate is compared. In other methods, the cell expresses anexpression profile gene set of at least one expression profile gene, andthe effect of the drug candidate on the expression of the set isdetermined. In some such methods, the profile gene set comprises atolerance set comprising carb anh II, IgD, CD72, SATBI, ApoE, CD83,cyclin D2, Cctq, MEF-2C, TGIF, Aeg-2, Egr-1, 1ck, Egr-2, E2-20K, pcp-4,kappa V, neurogranin, NAB2, gfi-l hIP-30, TRAP, bmk, CD36, Evi-2,vimetin, Ly6E.1, and c-fes. In some such methods, the expression ofhIP-30, TRAP, bmk, CD36, Evi-2, and c-fes are decreased and theexpression of carb anh II, CD72, SATB 1, ApoE, CD83, cyclin D2, Cctq,MEF-2C, TGIF, Aeg-2, Egr-1, 1ck, Egr-2, E2-20K, pcp-4, kappa V,neurogranin, NAB2, gfi-1 are increased as a result of the introductionof the drug candidate.

[0009] In some methods, the set comprises a stimulation set comprisingEgr-1, Egr-2, NAB2, mafK, LRG-21, c-fos, c-myc, Stra13, AhR, gadd153,C/EBP beta, TIS11b, TIS11, gfi-1, EZF, Nur77, LSIRF, SNK, PAC-1,kir/gem, MacMARCKS, PEP, MKP 1, hRab30, MIP-1b, MIP-1a, EB12, BL34,IL1-R2, TIS7, MyD116, A1, UPAR, RP105, Evi2 4F2, CD72, Id3, BKLF, LKLF,EFP, Stat1, bcl-3, hRhoH, TRAF5, SLAP, LT-beta, IFNg-RII, GILZ, Caspase2, gadd45, CDC47, NAG, scd2, kappa 0 ig, B29, iap38, G7e, and hIFI-204.In some such methods, the expression of Id3, BKLF, LKLF, EFP, Stat1,bcl-3, hRhoH, TRAF5, SLAP, LT-beta, IFNg-RII, GILZ. Caspase 2, gadd45,CDC47, NAG, scd2, kappa 0 ig, B29, iap38, G7e, and hIFI-204 aredecreased and the expression of Egr-1, Egr-2, NAB2, mafK, LRG-21, c-fos,c-myc, Stra13, AhR, gadd153, C/EBP beta, TIS1 lb, TISI1, gfi-1, EZF,Nur77, LSIRF, SNK, PAC-1, kir/gem, MacMARCKS, PEP, MKP1, hRab30, MIP-1b,MIP-1a, EB12, BL34, IL1-R2, TIS7, MyD116, A1, UPAR, RP105, Evi-24F2,CD72 are increased as a result of the introduction of the drugcandidate.

[0010] In some methods, the set comprises an immunosuppression setcomprising hIFI-204, hRhoH, caspase 2, B29, SLAP, NAG, iap38, gadd45,BKLF, G7e, Id3, scd2, GILZ, Stat1, kappa 0 ig, LT-beta, LKLF, IFNg-RII,mCDC47, EFP, TRAF5, and bc1-3. In some such methods, theimmunosuppressive set further comprises c-fos, gadd153, EZF, C/EBP beta,Stra13, NAB2, mafK, and LRG-21. In some such methods the expression ofc-fos, gadd153, EZF, C/EBP beta, Stra13, NAB2, mafK, and LRG-21 areincreased as a result of the introduction of the drug candidate. In somemethods the expression of hIFI-204, hRhoH, caspase 2, B29, SLAP, NAG,iap38, gadd45, BKLF, G7e, Id3, scd2, GILZ, Statl, kappa 0 ig, LT-beta,LKLF, IFNg-RII, mCDC47, EFP, TRAF5, and bcl-3 are decreased and theexpression of LSIRF, kir/gem, MKP1, hRab30, AhR, c-myc, Il1-R2,TIS11b,Evi-2, A1, EB12, MyD116, MacMARCKS, MIP-1b, MIP-1a, PEP, CD72 areincreased as a result of the introduction of the drug candidate.

[0011] The invention further provides methods of screening for abioactive agent capable of binding to a B lymphocyte modulator protein(BLMP). The BLMP and a candidate bioactive agent are combined. Thebinding of the candidate agent to the BLMP is then determined. In somesuch methods, the BLMP is selected from the group consisting of Egr-1,Egr-2, Nur77, c-myc, MIP-1a, MIP-1b,BL34, gfi-1, NAB2, neurogranin,SLAP, A1, E2-20K, SATB I, Cctq, kappa V, pcp-4, TGIF, CD83, ApoE, Aeg-2,CD72, cyclin D2, 1ck, MEF-2C, bmk, IgD, Evi-2, vimentin, CD36, c-fes,c-fos, TRAP, hIP30, Ly6E.1, LRG-21, Fos B, gadd153, mafK, Ah-R, C/EBPbeta, EZF, TIS7, TIS11, TIS11b, LSIRF, MKP1, PAC-1, PEP, MacMARCKS, SNK,Stra13, kir/gem, EB12, IL1-R2, MyD116, RP105, uPAR, 4F2, hRab30, Jd3,BKLF, LKLF, EFP, bcl-3, caspase 2, GILZ, hIFI-204, hRhoH, TRAF5,LT-beta, IFNg-RII, gadd45, CDC47, NAG, scd2, kappa 0 ig, iap38, G7e,B29, and SCD2.

[0012] The invention further provides methods for screening for abioactive agent capable of modulating the activity of a B lymphocytemodulator protein (BLMP). The BLMP and a candidate bioactive agent arecombined. The effect of the candidate agent on the bioactivity of theBLMP is then determined.

[0013] In some such methods the BLMP is selected from the groupconsisting of Egr-1, Egr-2, Nur77, c-myc, MIP-1a, MIP-1b,BL34, gfi-1,NAB2, neurogranin, SLAP, A1, E2-20K, SATB1, Cctq, kappa V, pcp-4, TGIF,CD83, ApoE, Aeg-2, CD72, cyclin D2, 1ck, MEF-2C, bmk, IgD, Evi-2,vimentin, CD36, c-fes, c-fos, TRAP, hIP30, Ly6E.1, LRG-21, Fos B,gadd153, mafK, Ah-R, C/EBP beta, EZF, TIS7, TIS 11, TIS11b, LSIRF, MKP1,PAC-1, PEP, MacMARCKS, SNK, Stra13, kir/gem, EB12, IL1-R2, MyD116,RP105, uPAR, 4F2, hRab30, Id3, BKLF, LKLF, EFP, bcl-3, caspase 2, GILZ,hIFI-204, hRhoH, TRAF5, LT-beta, IFNg-RII, gadd45, CDC47, NAG, scd2,kappa 0 ig, iap38, G7e, B29, and SCD2.

[0014] The invention further provides a method of evaluating the effectof an immunosuppressive drug. In such methods, the drug is administeredto a patient; b) a cell sample is removed from the patient; and c) theexpression profile of the cell sample is determined. Some such methodsfurther comprise comparing the expression profile to an expressionprofile of a healthy individual. In some such methods the expressionprofile includes at least one gene selected from the group consisting ofEgr-1, Egr-2, Nur77, c-myc, MIP-1a, MIP-1b,BL34, gfi-1, NAB2,neurogranin, SLAP, A1, E2-20K, SATBI, Cctq, kappa V, pcp-4, TGIF, CD83,ApoE, Aeg-2, CD72, cyclin D2, 1ck, MEF-2C, bmk, IgD, Evi-2, vimentin,CD36, c-fes, c-fos, TRAP, hIP30, Ly6E.1, LRG-21, Fos B, gadd153, mafk,Ah-R, C/EBP beta, EZF, TIS7, TIS11, TIS11b, LSIRF, MKP1, PAC-1, PEP,MacMARCKS, SNK, Stra13, kir/gem, EB12, IL1-R2, MyD116, RP105, uPAR, 4F2,hRab30, Id3, BKLF, LKLF, EFP, bcl-3, caspase 2, GILZ, hIFI-204, hRhoH,TRAF5, LT-beta, IFNg-RII, gadd45, CDC47, NAG, scd2, kappa 0 ig, iap38,G7e, B29, and SCD2.

[0015] The invention further provides an array of probes. The arraycomprises a support bearing a plurality of nucleic acid probescomplementary to a plurality of mRNAs fewer than 1000 in number, whereinthe plurality of mRNA probes includes an mRNA expressed by a geneselected from the group consisting of Egr-1, Egr-2, Nur77, c-myc,MIP-1a, MIP-1b,BL34, gfi-1, NAB2, neurogranin, SLAP, A1, E2-20K, SATB1,Cctq, kappa V, pcp-4, TGIF, CD83, ApoE, Aeg-2, CD72, cyclin D2, 1ck,MEF-2C, bmk, IgD, Evi-2, vimentin, CD36, c-fes, c-fos, TRAP, hIP30,Ly6E.1, LRG-21, Fos B, gadd153, mafK, Ah-R, C/EBP beta, EZF, TIS7, TISI1, TIS1 lb, LSIRF, MKP1, PAC-1, PEP, MacMARCKS, SNK, Stra13, kir/gem,EB12, IL1-R2, MyD116, RP105, UPAR, 4F2, hRab30, Id3, BKLF, LKLF, EFP,bcl-3, caspase 2, GILZ, hIFI-204, hRhoH, TRAF5, LT-beta, IFNg-RII,gadd45, CDC47, NAG, scd2, kappa 0 ig, iap38, G7e, B29, and SCD2. Somesuch arrays comprise a plurality of sets of probes wherein each set ofprobes iscomplementary to subsequences from a mRNA. In some arrays theprobes are cDNA sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1. Gene expression changes in B lymphocytes responding toforeign antigen. A. Genes with increased mRNA levels after 1 hrstimulation. 37 genes that showed significantly (p<0.00018) increasedexpression (see methods) and showed a median fold change of >1.75 weresorted by putative function. (CD72 is also shown but only increased 1.5fold. BL34 is represented twice on the arrays, both sets of data areshown.) Each line represents one experiment. The left end of the lineshows hybridization intensity in resting B cells mock stimulated inmedium alone for one hour, the right end of the line shows intensity inB cells stimulated for one hour through the antigen receptor. Of theseven experiments shown, 3 experiments were with Ig^(HEL) transgenic Bcells stimulated with medium alone or with HEL, and 4 experiments werewith non-transgenic B cells stimulated with medium alone or withanti-mu. Analysis of variance showed that the basal profiles andresponses to stimulation for Ig^(HEL) and non-transgenic B cells wereessentially identical and the results have been presented together forclarity. Spiking known concentrations of bacterial transcripts allows anapproximate calibration of 5 intensity units/copy/cell assuming 300, 000transcripts per cell. B. Genes with decreased mRNA levels after 1 hrstimulation. Hybridization intensities are represented as for FIG. 1A.(GILZ is represented twice on the arrays, both sets of data are shown.).C. 1 and 6 hr timepoints of transcripts increased at 1 hr. Results arefrom 2 experiments showing HEL stimulation of Ig^(HEL) transgenic Bcells. Each experiment is represented by a line. The left end of theline is the intensity of the transcript in B cells mock stimulated for 1hr, the middle of the line is the intensity after 1 hr stimulation withHEL, the end of the line is the intensity after 6 hr stimulation withHEL. Genes are shown in order of exaggerated, sustained and transientincreases relative to mock and 1 hr stimulated samples. D. 1 and 6 hrtimepoints of transcripts decreased at 1 hr. Results are from 2experiments with HEL stimulation of Ig transgenic B cells and arerepresented as in FIG. 1C.

[0017]FIG. 2. Gene expression changes in B lymphocytes responding toself antigen. A. Genes upregulated in tolerant cells compared to naivecells. The left end of each line represents hybridization level in naïveIg^(HEL) cells, the right end of the line represents hybridization levelin tolerant sHEL/Ig^(HEL) cells. Data points that are joined are fromseparate cell populations from genetically distinct animals—each linerepresents samples prepared in parallel on the same day. Five sets ofdata were derived from negatively depleted B cell preparations and twosets from FACS-sorted cells. One set of preparations included twotolerant cell samples and one naive cell sample. This set is representedas 2 lines joining the naive cell hybridization intensity to each of thetolerant cell intensities. B. Genes downregulated in tolerant cellscompared to naive cells. Data is represented as in FIG. 2A.

[0018]FIG. 3. Gene expression changes in B lymphocytes responding toforeign antigen in the presence of FK506 or PD98059. A. FK506sensitivity of the 1 hr upregulated genes defined in FIG. 1. B cellswere stimulated in the presence or absence of FK506, or were mockstimulated. Data are shown from 5 experiments and genes are shown inincreasing order of median FK506 sensitivity. Each line represents oneexperiment. The left end of the line is hybridization intensity inresting B cells, the middle of the line is intensity in B cellsstimulated for one hour through the antigen receptor and the right endof the line is intensity in B cells stimulated for one hour in thepresence of FK506. Of the five experiments shown, 3 experiments werewith IgHEL transgenic B cells stimulated with medium, HEL or HEL/FK506,and 2 experiments were with non-transgenic B cells stimulated withmedium, anti-mu or anti-mu/FK506. B. FK506 sensitivity of the 1 hrdown-regulated genes. Data is represented as for FIG. 3A. C. Correlationbetween sensitivity to FK506 and sensitivity to EGTA for antigen-inducedtranscripts. For the 37 induced genes defined in FIG. 1A, the relativeinduction in the presence of EGTA was calculated as average(antigen/EGTA-mock)/(antigen-mock), in two experiments with IgHELtransgenic cells stimulated with HEL. For the same transcripts, relativeinduction in the presence of FK506 was calculated as median of(antigen/FK506-mock)/(antigen-mock) over 5 experiments. D. Upper twopanels: upregulation of Egr-1 by anti-mu stimulation of non-transgenic Bcells is inhibited by PD98059 with an IC50 of ˜5 gM. Regulation of other1 hour response genes is less sensitive to PD98059. Lower panel: 3transcripts upregulated by both foreign and self antigen are sensitiveto PD98059. Left most four columns for each gene represent data fromnon-transgenic B cells stimulated with anti-mu, right most 3 columnsrepresent data from Ig^(HEL) transgenic B cells stimulated with HEL.

[0019]FIG. 4A. Summary table of biochemical pathways in tolerant cellsand naive cells exposed to foreign antigen in the presence or absence ofFK506 and PD98059. B. Potential mechanisms of tolerance, immunity andimmunosuppression suggested by the gene expression analysis. Font sizereflects mRNA or protein expression level relative to mock stimulatedcells (immunosuppression and activation panels) or naive cells(tolerance panel). Tolerant cells have decreased surface IgM (sIgM) butincreased IgD (mRNA and protein): sIg engagement by self-antigen causesdecreased tyrosine phosphorylation relative to activated cells. Proximalsignaling from sIg can be modulated in activated and tolerant cells byrecruitment of SHP1 by increased CD72. Activation of naive cells causesa robust calcium flux that triggers NFKB, JNK and NFAT. All thesepathways are blocked by FK506 through inhibition of calcineurin:calmodulin action can be regulated in naive and immunosuppressed cellsby neurogranin and in tolerant cells by neurogranin and pcp-4. Egrfamily transcription is predicted to be different under the 3conditions: in activated cells both Egr-1 and Egr-2 are upregulatedpreceding upregulation of NAB2; in immunosuppressed cells, only Egr-1 isupregulated; and in tolerant cells Egr-1 and Egr-2 are only weaklyupregulated and can have different effects on transcription in thepresence of increased NAB2. The balance between mitosis and apoptosis islikely to be in part determined by upregulation of the proto-oncogenesc-myc and LSIRF and the anti-apoptotic gene Al in activated cells. Thesechanges are blocked by tolerance and partially blocked by FK506.Downregulation of LKLF, which is sufficient to cause T cell activation,is partially inhibited by FK506 and is blocked in tolerance.Upregulation of surface activation markers CD69 and B7.2 is uninhibitedby FK506 but is blocked in tolerance. The level of B7.2 on B cells iscritical in interaction with antigen specific T cells.

DETAILED DESCRIPTION

[0020] 1. Definitions

[0021] The term patient includes mammals, such as humans, domesticanimals (e.g., dogs or cats), farm animals (cattle, horses, or pigs),monkeys, rabbits, rats, mice, and other laboratory animals.

[0022] The terms “nucleic acid” or “nucleic acid molecule” refer to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, can encompass knownanalogs of natural nucleotides that can fimction in a similar manner asnaturally occurring nucleotides.

[0023] A polynucleotide probe is a single stranded nucleic acid capableof binding to a target nucleic acid of complementary sequence throughone or more types of chemical bonds, usually through complementary basepairing, usually through hydrogen bond formation. A polynucleotide probecan include natural (i.e., A, G, C, or T) or modified bases (e.g.,7-deazaguanosine, inosine). Therefore, polynucleotide probes can5-10,000, 10-5,000, 10-500, 10-50, 10-25, 10-20, 15-25, and 15-20 baseslong. Probes are typically about 10-50 bases long, and are often 15-20bases. In its simplest embodiment, the array includes test probes (alsoreferred to as polynucleotide probes) more than 5 bases long, preferablymore than 10 bases long, and some more than 40 bases long. The probescan also be less than 50 bases long. In some cases, these polynucleotideprobes can range from about 5 to about 45 or 5 to about 50 nucleotideslong, or from about 10 to about 40 nucleotides long, or from about 15 toabout 40 nucleotides in length. The probes can also be about 20 or 25nucleotides in length.

[0024] In addition, the bases in a polynucleotide probe can be joined bya linkage other than a phosphodiester bond, so long as it does notinterfere with hybridization. Thus, polynucleotide probes can be peptidenucleic acids in which the constituent bases are joined by peptide bondsrather than phosphodiester linkages. The length of probes used ascomponents of pools for hybridization to distal segments of a targetsequence often increases as the spacing of the segments increasedthereby allowing hybridization to be conducted under greater stringencyto increase discrimination between matched and mismatched pools ofprobes.

[0025] Relatively short polynucleotide probes can be sufficient tospecifically hybridize to and distinguish target sequences. Therefore,the polynucleotide probes can be less than 50 nucleotides in length,generally less than 46 nucleotides, more generally less than 41nucleotides, most generally less than 36 nucleotides, preferably lessthan 31 nucleotides, more preferably less than 26 nucleotides, and mostpreferably less than 21 nucleotides in length. The probes can also beless than 16 nucleotides, less than 13 nucleotides in length, less than9 nucleotides in length and less than 7 nucleotides in length.

[0026] Typically, arrays can have polynucleotides as short as 10nucleotides or 15 nucleotides. In addition, 20 or 25 nucleotides can beused to specifically detect and quantify nucleic acid expression levels.Where ligation discrimination methods are used, the polynucleotidearrays can contain shorter polynucleotides. Arrays containing longerpolynucleotides are also suitable. High density arrays can comprisegreater than about 100, 1000, 16,000, 65,000, 250,000 or even greaterthan about 1,000,000 different polynucleotide probes.

[0027] The term “target nucleic acid” refers to a nucleic acid (oftenderived from a biological sample), to which the polynucleotide probe isdesigned to specifically hybridize. It is either the presence or absenceof the target nucleic acid that is to be detected, or the amount of thetarget nucleic acid that is to be quantified. The target nucleic acidhas a sequence that is complementary to the nucleic acid sequence of thecorresponding probe directed to the target. The term target nucleic acidcan refer to the specific subsequence of a larger nucleic acid to whichthe probe is directed or to the overall sequence (e.g., gene or mRNA)whose expression level it is desired to detect. The difference in usagecan be apparent from context.

[0028] “Subsequence” refers to a sequence of nucleic acids that comprisea part of a longer sequence of nucleic acids.

[0029] “Gene” refers to a unit of inheritable genetic material found ina chromosome, such as in a human chromosome. Each gene is composed of alinear chain of deoxyribonucleotides which can be referred to by thesequence of nucleotides forming the chain. Thus, “sequence” is used toindicate both the ordered listing of the nucleotides which form thechain, and the chain which has that sequence of nucleotides. The term“sequence” is used in the same way in referring to RNA chains, linearchains made of ribonucleotides. The gene includes regulatory and controlsequences, sequences which can be transcribed into an RNA molecule, andcan contain sequences with unknown function. Some of the RNA products(products of transcription from DNA) are messenger RNAs (mRNAs) whichinitially include ribonucleotide sequences (or sequence) which aretranslated into a polypeptide and ribonucleotide sequences which are nottranslated. The sequences which are not translated include controlsequences, introns and sequences with unknowns function. It can berecognized that small differences in nucleotide sequence for the samegene can exist between different persons, or between normal cells andcancerous cells, without altering the identity of the gene.

[0030] “Gene expression pattern” means the set of genes of a specifictissue or cell type that are transcribed or “expressed” to form RNAmolecules. Which genes are expressed in a specific cell line or tissuecan depend on factors such as tissue or cell type, stage of developmentor the cell, tissue, or target organism and whether the cells are normalor transformed cells, such as cancerous cells. For example, a gene canbe expressed at the embryonic or fetal stage in the development of aspecific target organism and then become non-expressed as the targetorganism matures. Alternatively, a gene can be expressed in liver tissuebut not in brain tissue of an adult human.

[0031] Specific hybridization refers to the binding, duplexing, orhybridizing of a molecule only to a particular nucleotide sequence understringent conditions when that sequence is present in a complex mixture(e.g., total cellular) DNA or RNA. Stringent conditions are conditionsunder which a probe can hybridize to its target subsequence, but to noother sequences. Stringent conditions are sequence-dependent and aredifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength, pH, and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. (As the targetsequences are generally present in excess, at T_(m), 50% of the probesare occupied at equilibrium). Typically, stringent conditions include asalt concentration of at least about 0.01 to 1.0 M Na ion concentration(or other salts) at pH 7.0 to 8.3 and the temperature is at least about30° C. for short probes (e.g., 10 to 50 nucleotides). Stringentconditions can also be achieved with the addition of destabilizingagents such as formamide or tetraalkyl ammonium salts. For example,conditions of 5×SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH7.4) and a temperature of 25-30° C. are suitable for allele-specificprobe hybridizations. (See Sambrook et al., Molecular Cloning 1989).

[0032] Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

[0033] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Defaultprogram parameters are used. Methods of alignment of sequences forcomparison are well-known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds 1995 supplement)).

[0034] One example of a useful algorithm is PILEUP. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). UsingPILEUP, a reference sequence is compared to other test sequences todetermine the percent sequence identity relationship using the followingparameters: default gap weight (3.00), default gap length weight (0.10),and weighted end gaps. PILEUP can be obtained from the GCG sequenceanalysis software package, e.g., version 7.0 (Devereaux et al., Nuc.Acids Res. 12:387-395 (1984).

[0035] Another example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST and theBLAST 2.0 algorithm, which are described in Altschul et al., J. Mol.Biol. 215:403-410 (1990) and Altschul et al., Nucleic Acids Res.25:3389-3402 (1977)). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). The BLASTN program (for nucleotidesequences) uses as defaults a word length (W) of 11, alignments (B) of50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.The BLASTP program (for amino acid sequences) uses as defaults a wordlength (W) of 3, and expectation (E) of 10, and the BLOSLUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)).

[0036] The term antibody is used to include intact antibodies andbinding fragments thereof. Typically, fragments compete with the intactantibody from which they were derived and with other antibodies forspecific binding to an antigen. The term antibody includes polyclonalantibodies, monoclonal antibodies, chimeric antibodies and humanizedantibodies, produced by immunization, from hybridomas, or recombinantly.

[0037] The term molecule is used broadly to mean an organic or inorganicchemical such as a drug; a peptide, including a variant or modifiedpeptide or peptide-like substance such as a peptidomimetic or peptoid;or a protein such as an antibody or a growth factor receptor or afragment thereof, such as an F_(v), F_(c) or F_(ab) fragment of anantibody, which contains a binding domain. A molecule can benonnaturally occurring, produced as a result of in vitro methods, or canbe naturally occurring, such as a protein or fragment thereof expressedfrom a cDNA library.

[0038] The term specific binding (and equivalent phrases) refers to theability of a binding moiety (e.g., a receptor, antibody, ligand orantiligand) to bind preferentially to a particular target molecule(e.g., ligand or antigen) in the presence of a heterogeneous populationof proteins and other biologics (i.e., without significant binding toother components present in a test sample). Typically, specific bindingbetween two entities, such as a ligand and a receptor, means a bindingaffinity of at least about 106 M⁻¹, and preferably at least about 10⁷,10⁸, 10⁹, or 10¹⁰ M⁻¹. In some embodiments specific (or selective)binding is assayed (and specific binding molecules identified) accordingto the method of U.S. Pat. No. 5,622,699; this reference and allreferences cited therein are incorporated herein by reference. Typicallya specific or selective reaction according to this assay is at leastabout twice background signal or noise and more typically at least about5 or at least about 100 times background, or more.

[0039] When the binding moiety is an antibody, a variety of immunoassayformats can be used to select antibodies that are specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with an antigen. See Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity (this reference andreferences cited therein are incorporated herein by reference).

[0040] The term “autoimmune disease” refers to a spontaneous or inducedmalfunction of the immune system of mammals in which the immune systemfails to distinguish between foreign immunogenic substances within themammal and/or autologous (“self”) substances and, as a result, treatsautologous (“self”) tissues and substances as if they were foreign andmounts an immune response against them. Autoimmune disease ischaracterized by production of either antibodies that react with selftissue, and/or the activation of immune effector T cells that areautoreactive to endogenous self antigens. Three main immunopathologicmechanisms act to mediate autoimmune diseases: 1) autoantibodies aredirected against functional cellular receptors or other cell surfacemolecules, and either stimulate or inhibit specialized cellular functionwith or without destruction of cells or tissues; 2)autoantigen—autoantibody immune complexes form in intercellular fluidsor in the general circulation and ultimately mediate tissue damage; and3) lymphocytes produce tissue lesions by release of cytokines or byattracting other destructive inflammatory cell types to the lesions.These inflammatory cells in turn lead to production of lipid mediatorsand cytokines with associated inflammatory disease.

[0041] The term “inflammation” refers to both acute responses (i.e.,responses in which the inflammatory processes are active) and chronicresponses (i.e., responses marked by slow progression and formation ofnew connective tissue). Acute and chronic inflammation may bedistinguished by the cell types involved. Acute inflammation ofteninvolves polymorphonuclear neutrophils; whereas chronic inflammation isnormally characterized by a lymphohistiocytic and/or granulomatousresponse. Inflammation includes reactions of both the specific andnon-specific defense systems. A specific defense system reaction is aspecific immune system reaction response to an antigen (possiblyincluding an autoantigen). A non-specific defense system reaction is aninflammatory response mediated by leukocytes incapable of immunologicalmemory. Such cells include granulocytes, macrophages, neutrophils andeosinophils. Examples of specific types of inflammation are diffuseinflammation, focal inflammation, croupous inflammation, interstitialinflammation, obliterative inflammation, parenchymatous inflammation,reactive inflammation, specific inflammation, toxic inflammation andtraumatic inflammation.

[0042] The term “immune-mediated” refers to a process that is eitherautoimmune or inflammatory in nature.

[0043] The term “perfect match probe” refers to a probe that has asequence that is perfectly complementary to a particular targetsequence. The test probe is typically perfectly complementary to aportion (subsequence) of the target sequence. The perfect match (PM)probe can be a “test probe,” a “normalization control” probe, anexpression level control probe and the like. A perfect match control orperfect match probe is, however, distinguished from a “mismatch control”or “mismatch probe.”

[0044] The term “mismatch control” or “mismatch probe” refer to probeswhose sequence is deliberately selected not to be perfectlycomplementary to a particular target sequence. For each mismatch (MM)control in a high-density array there typically exists a correspondingperfect match (PM) probe that is perfectly complementary to the sameparticular target sequence. The mismatch can comprise one or more bases.While the mismatches) can be located anywhere in the mismatch probe,terminal mismatches are less desirable as terminal mismatch is lesslikely to prevent hybridization of the target sequence.

[0045] The term “probe set” comprises at least a plurality of genesperfectly matched with a known target sequence.

[0046] The terms “background” or “background signal intensity” refer tohybridization signals resulting from non-specific binding, or otherinteractions, between the labeled target nucleic acids and components ofthe polynucleotide array (e.g., the polynucleotide probes, controlprobes, or the array substrate). Background signals can also be producedby intrinsic fluorescence of the array components themselves. A singlebackground signal can be calculated for the entire array, or a differentbackground signal can be calculated for each region of the array. Insome embodiments, background is calculated as the average hybridizationsignal intensity for the lowest 1% to 10% of the probes in the array, orregion of the array. In expression monitoring arrays (i.e., where probesare preselected to hybridize to specific nucleic acids (genes), adifferent background signal can be calculated for each target nucleicacid. Where a different background signal is calculated for each targetgene, the background signal is calculated for the lowest 1% to 10% ofthe probes for each gene. Where the probes to a particular genehybridize well and thus appear to be specifically binding to a targetsequence, they should not be used in a background signal calculation.Alternatively, background can be calculated as the average hybridizationsignal intensity produced by hybridization to probes that are notcomplementary to any sequence found in the sample (e.g., probes directedto nucleic acids of the opposite sense or to genes not found in thesample such as bacterial genes where the sample is of mammalian origin).Background can also be calculated as the average signal intensityproduced by regions of the array that lack any probes at all.

[0047] The term “quantifying” when used in the context of quantifyingnucleic acid abundance or concentrations (e.g., transcription levels ofa gene) can refer to absolute or to relative quantification. Absolutequantification can be accomplished by inclusion of knownconcentration(s) of one or more target nucleic acids (e.g., controlnucleic acids such as BioB or with known amounts the target nucleicacids themselves) and referencing the hybridization intensity ofunknowns with the known target nucleic acids (e.g., through generationof a standard curve). Alternatively, relative quantification can beaccomplished by comparison of hybridization signals between two or moregenes, or between two or more treatments to quantify the changes inhybridization intensity and, by implication, transcription level.

[0048] The term “cluster” or “clustering” refers to clusteringalgorithms, such as principal components analysis and variableclustering analysis. These algorithms serve to “cluster” cells intogroups. The purpose of clustering is to place the isolates into groupsor clusters suggested by the data, not defined a priori, such thatisolates in a given cluster tend to be similar and isolates in differentclusters tend to be dissimilar. Methods of clustering are described inTamayo et al., Proc. Natl. Acad. Sci U.S.A. (1999) 96: 2907-2912.

[0049] 2. Gene Expression Profiles

[0050] The present invention provides novel methods for screening forcompositions which modulate B cell activity. The expression levels ofgenes are determined for different cellular states of B cells to provideexpression profiles. A B cell expression profile of a particular B cellstate can be a “fingerprint” of the state; while two states can have anyparticular gene similarly expressed, the evaluation of a number of genessimultaneously allows the generation of a gene expression profile thatis unique to the state of the cell. By comparing expression profiles ofB cells in naive, activated, immunosuppressed, tolerant or restingstates, information regarding which genes are important (including bothup- and down-regulation of genes) in each of these states is obtained.This information can then be used in a number of ways. For example, theevaluation of a particular treatment regime can be evaluated: does animmunosuppressive drug act like an immunosuppressive drug in thisparticular patient. Similarly, diagnosis can be done or confirmed: doesthis patient have the gene expression profile of immunosuppressed Bcells. Furthermore, these gene expression profiles can be used in drugcandidate screening to find drugs that mimic a particular expressionprofile; for example, screening can be done for drugs that induce B celltolerance as evidenced by a tolerant expression profile. Accordingly,genes are identified and described which are differentially expressedwithin and among B cells in different states, from which the expressionprofiles are generated as further described herein. For example,determinations of differentially expressed nucleic acids are providedherein for B cells which are resting, activated, immunosuppressed,naive, and tolerant.

[0051] “Differential expression,” or grammatical equivalents as usedherein, refers to both qualitative as well as quantitative differencesin the genes' temporal and/or cellular expression patterns within andamong B cells. Thus, a differentially expressed gene can qualitativelyhave its expression altered, including an activation or inactivation in,for example, tolerant versus immunosuppressed cells, rested, naive oractivated cells, or in a healthy B cell response versus an abnormal Bcell response. Genes can be turned on or turned off in a particularstate, relative to another state. Any comparison of two or more statescan be made. Such a qualitatively regulated gene will exhibit anexpression pattern within a state or cell type which can be detectableby standard techniques in one such state or cell type, but can be notdetectable in both. Alternatively, the determination can be quantitativein that expression is increased or decreased; that is, the expression ofthe gene is either upregulated, resulting in an increased amount oftranscript, or downregulated, resulting in a decreased amount oftranscript. The degree to which expression differs need only be largeenough to quantify using standard characterization techniques, forexample, by using Affymetrix GeneChip™ expression arrays (Lockhart,Nature Biotechnology, (1996) 14:1675-1680; this reference and allreferences cited therein are incorporated by reference). Other methodsinclude, but are not limited to, quantitative reverse transcriptase PCR,Northern analysis and RNase protection. Preferably the change ormodulation in expression (i.e., upregulation or downregulation) is atleast about 5%, more preferably at least about 10%, more preferably, atleast about 20%, more preferably, at least about 30%, or more preferablyby at least about 50%, or at least about 75%, and more preferably atleast about 90%.

[0052] Any one, two, three, four, five, or ten or more genes can beevaluated. These genes include, but are not limited to, Egr-1, Egr-2,Nur77, c-myc, MIP-1a, MIP-1b, BL34, gfi-1, NAB2, neurogranin, SLAP, A1,E2-20K, SATB1, Cctq, kappa V, pcp-4, TGIF, CD83, ApoE, Aeg-2, CD72,cyclin D2, 1ck, MEF-2C, bmk, IgD, Evi-2, vimentin, CD36, c-fes, c-fos,TRAP, hIP30, Ly6E.1, LRG-21, Fos B, gadd153, mafk, Ah-R, C/EBP beta,EZF, TIS7, TIS11, TIS11b, LSIRF, MKP1, PAC-1, PEP, MacMARCKS, SNK,Stra13, kir/gem, EB12, IL1-R2, MyD116, RP105, uPAR, 4F2, hRab30, Id3,BKLF, LKLF, EFP, bcl-3, caspase 2, GILZ, hIFI-204, hRhoH, TRAF5,LT-beta, IFNg-RII, gadd45, CDC47, NAG, scd2, kappa 0 ig, iap38, G7e,B29, and SCD2 (the accession numbers for these genes can be found inTable 1). Generally, oligonucleotide sequences used in the evaluation ofthese genes are derived from their 3′ untranslated regions.

[0053] Differentially expressed genes can represent “expression profilegenes”, which includes “target genes”. “Expression profile gene,” asused herein, refers to a differentially expressed gene whose expressionpattern can be used in methods for identifying compounds useful in themodulation of B cell states or activity, or the treatment of disorders,or alternatively, the gene can be used as part of a prognostic ordiagnostic evaluation of immune disorders. For example, the effect ofthe compound on the expression profile gene normnally displayed inconnection with a particular state, such as tolerance, for example, canbe used to evaluate the efficacy of the compound to modulate that state,or preferably, to induce or maintain that state. Such assays are furtherdescribed below. Alternatively, the gene can be used as a diagnostic orin the treatment of immune disorders as also further described below. Insome instances, only a fragment of an expression profile gene is used,as further described below.

[0054] “Expression profile,” as used herein, refers to the pattern ofgene expression generated from two up to all of the expression profilegenes which exist for a given state. As outlined above, an expressionprofile is in a sense a “fingerprint” or “blueprint” of a particularcellular state; while two or more states have genes that are similarlyexpressed, the total expression profile of the state will be unique tothat state. The gene expression profile obtained for a given B cellstate can be useful for a variety of applications, including diagnosisof a particular disease or condition and evaluation of various treatmentregimes. In addition, comparisons between the expression profiles ofdifferent B cell states can be similarly informative. An expressionprofile can include genes which do not appreciably change between twostates, so long as at least two genes which are differentially expressedare represented. The gene expression profile can also include at leastone target gene, as defined below. Alternatively, the profile caninclude all of the genes which represent one or more states. Specificexpression profiles are described below.

[0055] Gene expression profiles can be defined in several ways. Forexample, a gene expression profile can be the relative transcript levelof any number of particular set of genes. Alternatively, a geneexpression profile can be defined by comparing the level of expressionof a variety of genes in one state to the level of expression of thesame genes in another state. For example, genes can be eitherupregulated, downregulated, or remain substantially at the same level inboth states.

[0056] The expression profile for an activated B cell compared to anaive resting B cell following lymphocyte activation for one hour isshown in FIG. 1. Lymphocyte activation as used herein refers to theantigen induced progression of B cells from the GO phase to the Gi phaseof the cell cycle. FIG. 1A shows the following upregulated genes afterlymphocyte activation for 1 hour: Egr-1, Egr-2, NAB2, mafk, LRG-21, FosB. cfos, c-myc, Stra13, AhR, gadd153, C/EBP beta, TIS11, TIS11b, gfi-1,EZF, Nur77, LSIRF, SNK, PAC-1, kir/gem, MacMARCKS, PEP, MKP1, hRab30,MIP-1a, MIP-1b, EBI2, BL34, ILI-R2, TIS7, MyD116, A1, uPAR, RP105,Evi-2, 4F2 and CD72; these genes are referred to herein as upregulatedearly activation B cell expression profile genes. FIG. 1B shows thefollowing genes that are downregulated after lymphocyte activation forone hour: Id3, BKLF, LKLF, EFP, Stat1, bcl-3, hRhoH, TRAF5, SLAP,LT-beta, IFNg-RII, GILZ, Caspase 2, gadd45, mCDC47, NAG, scd2, kappa 0Ig, B29, iap38, G7e, and hIFI-204; these genes are referred to herein asdownregulated early activation B cell expression profile genes.

[0057] Also provided herein are gene expression profiles for tolerant Bcells compared to naive B cells after activation by self or foreignantigen. Tolerance is generally defined as a state of alteredresponsiveness to a particular antigen that prevents development ofeither a cellular- or antibody-based immune response to that antigen.FIG. 2A shows genes that are upregulated in tolerant cells compared tonaive cells after activation by self-antigen: IgD, carb anh II, CD72,SATB1, ApoE, CD83, cyclin D2, Cctq, MEF-2C, TGIF, Aeg-2, Egr-1, 1ck,Egr-2, E2-20K, pcp-4, kappa V, neurogranin, NAB2 and gfi-1. FIG. 2Bshows the following genes that are downregulated in tolerant cellscompared to naive cells after activation by self-antigen: Ly6E.1,vimentin, hIP-30, TRAP, bmk, CD36, Evi-2, and c-fes.

[0058] Also provided herein are gene expression profiles for B cellactivation inhibited by immunosuppressive agents, as generally outlinedbelow. Examples of immunosuppressive drugs which inhibit B cellactivation include FK506 (see, e.g., Wicker, L. S. et al., Eur J.Immunol (1990) 20: 2277-83) or cyclosporin A (see, e.g., Clin ImmunolImmunopathol (1996) 80(3 Pt 2): S40-5). As used herein,immunosuppression and tolerance include the suppression of B lymphocyteactivation. Agents which modulate immunosuppression are referred toherein as immunosuppressants, immunosuppressant modulators, orimmunosuppressive agents. The expression profile for immunosuppressed Bcells compared to activated and resting B cells is shown in FIG. 3. FIG.3A and FIG. 3B show the upregulated and downregulated early activation Bcell expression profile genes where each line individually shows onegene in the resting state, activated state and immunosuppressed state byreading the line left to right respectively. Thus, a gene sensitive toimmunosuppression is represented by a peak for upregulated genes (FIG.3A) and valleys for downregulated genes (see FIG. 3B). FIG. 3A shows theimmunosuppressive sensitivity of the following upregulated earlyactivation B cell expression profile genes in order of sensitivity,where the right side of FIG. 3A shows the most sensitive genes.“Sensitive” in this context means that gene expression is downregulatedas compared to the active state. Sensitive upregulated early activationB cell expression profile genes include: LSIRF, kir/gem, MKP1, hRab30,AhR, c-myc, ILIR2, TIS11b, Evi-2, A1, EB12, MyD116, MacMARCKS, MIP-1b,Egr-2, MIP-1a, PEP and CD72. Upregulated early activation B cellexpression profile genes that are less than 30% inhibited byimmunosuppressive agents include: c-fos, gadd153, EZF, C/EBP beta,Stra13, mafK, LRG-21, BL34, SNK, uPAR, TIS7, PAC-1, Fos B, TIS11, gfi-1,Egr-1, 4F2, RP 105 and Nur77.

[0059]FIG. 3B shows the immunosuppressive sensitivity of thedownregulated early activation B cell expression profile genes in orderof sensitivity, where the right side of FIG. 3B shows the most sensitivegenes. Sensitive downregulated early activation B cell expressionprofile genes include: LKLF, IFNg-RII, CDC47, EFP, TRAF5 and bcl-3.Downregulated early activation B cell expression profile genes that areless than 30% inhibited by immunosuppressive agents include: hIFI-204,hRhoH, caspase 2, B29, SLAP, NAG iap38, gadd45, BKLF, G7e, 1d3, scd2,GILZ, StatI, kappa 0 ig, and LT-beta.

[0060] A gene expression profile can include a combination of at leasttwo of Egr-1, Nur77, c-myc, MIP-1a, MIP-1b, BL34, gfi-1, NAB2,neurogranin, and SLAP. A1 can also be included in the expression profileof tolerant cells as shown in FIG. 4. Another target gene for toleranceis B7.2, which upregulation is inhibited in tolerance but not inimmunosuppression. A preferred target gene for tolerance or tolerancemodulation is NAB2 which is upregulated in tolerant B cells compared toresting or naive cells. Moreover, target genes for tolerance include:CD72, neurogranin, pcp-4, Egr-1, Egr-2, NAB2, myc, LSIRF, A1, and LKLFwhich are downregulated in tolerance; these changes appear to be uniqueto the tolerance phenotype and are not seen in response to an activatingsignal. Agents which modulate or induce a state of tolerance arereferred to herein as tolerants. In a preferred expression profile, thetotal expression profile is recreated by at least one small molecule(e.g., FK506 or cyclosporin A) or other pharmacological intervention. Inone embodimnent, at least one of or all of Egr-1, Egr-2, c-myc and c-fosare suppressed while NAB2 is upregulated.

[0061] 3. Target and Pathway Genes

[0062] In addition to expression profile genes, the present inventionalso provides target genes. “Target gene,” as used herein, refers to adifferentially expressed expression profile gene whose expression isunique for a particular state, such that the presence or absence of thetranscript of a target gene(s) can indicate the state the cell is in. Atarget gene can be completely unique to a particular state; the presenceor absence of the gene is only seen in a particular cell state, oralternatively, cells in all other states express the gene but it is notseen in the first state. Thus for example NAB2 is not expressed in naiveB cells but is expressed in all other states. Alternatively, targetgenes can be identified as relevant to a comparison of two states, thatis, the state is compared to another particular state or standard todetermine the uniqueness of the target gene. Target genes can be used inthe diagnostic, prognostic, and compound identification methodsdescribed herein.

[0063] It should be understood that a target gene for a first state canbe an expression profile gene for a second state. The presence orabsence of a particular target gene in one state can be diagnostic ofthe state; the same gene in a different state can be an expressionprofile gene.

[0064] Further, pathway genes are provided herein. “Pathway genes” aredefined by the ability of their gene products to interact withexpression profile genes. Pathway genes can also exhibit target geneand/or expression profile gene characteristics and can be included asmodulators of expression profile genes as further described below.

[0065] The present invention includes the products of such expressionprofile, target, and pathway genes, as well as antibodies to such geneproducts. Furthermore, the engineering and use of cell- and animal-basedmodels of B cell states to which such profiles, genes and gene productscan contribute, are also described.

[0066] 4. Sample Preparation

[0067] To measure the transcription level (and thereby the expressionlevel) of a gene or genes, a nucleic acid sample comprising mRNAtranscript(s) of the gene or genes, or nucleic acids derived from themRNA transcript(s) is provided. A nucleic acid derived from an mRNAtranscript refers to a nucleic acid for whose synthesis the mRNAtranscript or a subsequence thereof has ultimately served as a template.Thus, a cDNA reverse transcribed from an mRNA, an RNA transcribed fromthat cDNA, a DNA amplified from the cDNA, an RNA transcribed from theamplified DNA, are all derived from the mRNA transcript and detection ofsuch derived products is indicative of the presence and/or abundance ofthe original transcript in a sample. Thus, suitable samples include mRNAtranscripts of the gene or genes, cDNA reverse transcribed from themRNA, cRNA transcribed from the cDNA, DNA amplified from the genes, RNAtranscribed from amplified DNA, and the like.

[0068] In some methods, a nucleic acid sample is the total mRNA isolatedfrom a biological sample. The term “biological sample”, as used herein,refers to a sample obtained from an organism or from components (e.g.,cells) or an organism. The sample can be of any biological tissue orfluid. Frequently the sample is from a patient. Such samples includesputum, blood, blood cells (e.g., white cells), tissue or fine needlebiopsy samples, urine, peritoneal fluid, and fleural fluid, or cellstherefrom. Biological samples can also include sections of tissues suchas frozen sections taken for histological purposes. Often two samplesare provided for purposes of comparison. The samples can be, forexample, from different cell or tissue types, from different species,from different individuals in the same species or from the same originalsample subjected to two different treatments (e.g., drug-treated andcontrol).

[0069] 5. Method

[0070] (A) Generation of cDNAs

[0071] For example, methods of isolation and purification of nucleicacids are described in detail in WO 97/10365, WO 97/27317, Chapter 3 ofLaboratory Techniques in Biochemistry and Molecular Biology:Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic AcidPreparation, P. Tijssen, ed. Elsevier, N.Y. (1993) and Chapter 3 ofLaboratory Techniques in Biochemistry and Molecular Biology:Hybridization With Nucleic Acid Probes, Part 1. Theory and Nucleic AcidPreparation, P. Tijssen, ed. Elsevier, N.Y. (1993)).

[0072] The total nucleic acid can be isolated from a given sample using,for example, an acid quanidinium-phenol-choloroform extraction methodand poly A⁺ mRNA is isolated by oligo dT column chromatography or byusing (dT)n magnetic beads (see, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual (2^(nd) ed.), Vols 1-3, Cold Spring HarborLaboratory, (1989), or Current Protocols in Molecular Biology, F.Ausubel et al., ed., Breene Publishing and Wiley-Interscience, N.Y.(1987)).

[0073] The sample mRNA can be reverse transcribed with a reversetranscriptase and a primer consisting of oligo dT and a sequenceencoding the phage T7 promoter to provide single stranded DNA template.The second DNA strand is polymerized using a DNA polymerase. Methods ofin vitro polymerization are well known (see, e.g., Sambrook, supra) andthis particular method is described in detail by Van Gelder, et al.,Proc. Natl. Acad. Sci. U.S.A 87: 1663-1667 (1990) report that in vitroamplification according to this method preserves the relativefrequencies of the various RNA transcripts. Eberwine et al., Proc. Natl.Acad. Sci. U.S.A 89:3010-3014 provide a further protocol that uses tworound of amplification via in vitro transcription thereby permittingexpression monitoring. Eberwine et al describe another method ofamplification in Methods (1996) 10(3): 283-8. Another method ofamplification is described in Dixon et al, Nucleic Acids Res (1998)26(19): 4426-31. A still further method of amplification is theamplification method described in Dulac et al., Cell (1995) 83: 195-206.An alternative method of amplification is described in U.S. Ser. No.60/126,796 filed on Mar. 30, 1999, which is herein incorporated byreference.

[0074] After amplification, the nucleic acids are typically cleaved intosmaller fragments. Cleavage can be achieved by DNaseI digestion,restriction enzyme digestion, or sonication. Nucleic acids are typicallylabeled. Label can be introduced during amplification either by linkageto one of the primers or by one of the nucleotides being incorporated.Alternatively, labeling can be effected after amplification and cleavageby end-labeling. Detectable labels suitable for use in the presentinvention include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means; see WO 97/10365.

[0075] In general, nucleic acid probes comprising the expression profilegenes, including differentially expressed genes and target genes, can beattached to a solid support, generally in an array format, to allow forgene expression monitoring. “Gene” in this context includes full lengthgenes and fragments thereof, and can comprise either the coding strandor its complement, and can be a portion of a gene, a regulatorysequence, genomic DNA, cDNA, RNA including mRNA and rRNA.

[0076] In some cases, the differentially expressed nucleic acid can be afragment, or expressed sequence tag (EST). Once a differentiallyexpressed nucleic acid which is not a full length gene is identified, itcan be cloned and, if necessary, its constituent parts recombined toform an entire fall length or mature differentially expressed nucleicacid. Using methods described herein and known in the art, it can beused to identify the full length clone. Wherein the full length nucleicacid has a signal peptide and/or transmembrane region(s), it can bemodified to exclude one or more of these regions so as to encode apeptide in its mature soluble form. Once isolated from its naturalsource, e.g., contained within a plasmid or other vector or excisedtherefrom as a linear nucleic acid segment, the recombinantdifferentially expressed nucleic acid can be further-used as a probe toidentify and isolate other differentially expressed nucleic acid acids.It can also be used as a “precursor” nucleic acid to make modified orvariant differentially expressed nucleic acid acids and proteins. Wheretwo or more nucleic acids overlap, the overlapping portion(s) of one ofthe overlapping nucleic acids can be omitted and the nucleic acidscombined for example by ligation to form a longer linear differentiallyexpressed nucleic acid so as to, for example, encode the full length ormature peptide. The same applies to the amino acid sequences ofdifferentially expressed polypeptides in that they can be combined so asto form one contiguous peptide.

[0077] It should be noted that the nucleic acid probes used herein neednot be identical to the wild-type genes listed above. Nucleic acidshaving sequence identity with differentially expressed nucleic acidspreferably have about 65% or 75%, more preferably greater than about80%, even more preferably greater than about 85% and most preferablygreater than 90% sequence identity. In some embodiments the sequenceidentity will be as high as about 93 to 95 or 98%. Sequence identitywill be determined using standard techniques known in the art,including, but not limited to, the local sequence identity algorithm ofSmith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequenceidentity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Drive,Madison, Wis.), the Best Fit sequence program described by Devereux etal, Nucl. Acid Res. 12:387-395 (1984), preferably using the defaultsettings, or by inspection.

[0078] The PCR method of amplification is described in PCR Technology:Principles and Applications for DNA Amplification (ed. H. A. Erlich,Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods andApplications (eds. Innis, et al., Academic Press, San Diego, Calif.,1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert etal., PCR Methods and Applications 1, 17 (1991); PCR (eds. McPherson etal., IRL Press, Oxford); and U.S. Pat. No. 4,683,202 (each of which isincorporated by reference for all purposes). Nucleic acids in a targetsample are usually labeled in the course of amplification by inclusionof one or more labeled nucleotides in the amplification mix. Labels canalso be attached to amplification products after amplification e.g., byend-labeling. The amplification product can be RNA or DNA depending onthe enzyme and substrates used in the amplification reaction.

[0079] Other suitable amplification methods include the ligase chainreaction (LCR) (see Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988), transcription amplification (Kwoh et al.,Proc. Natl. Acad. Sci. U.S.A 86, 1173 (1989)), and self-sustainedsequence replication (Guatelli et al., Proc. Nat. Acad. Sci. U.S.A 87,1874 (1990)) and nucleic acid based sequence amplification (NASBA). Thelatter two amplification methods involve isothermal reactions based onisothermal transcription, which produce both single stranded RNA (ssRNA)and double stranded DNA (dsDNA) as the amplification products in a ratioof about 30 or 100 to 1, respectively.

[0080] A variety of labels can be incorporated into target nucleic acidsin the course of amplification or after amplification. Suitable labelsinclude fluorescein or biotin, the latter being detected by stainingwith phycoerythrin-streptavidin after hybridization. In some methods,hybridization of target nucleic acids is compared with control nucleicacids. Optionally, such hybridizations can be performed simultaneouslyusing different labels are used for target and control samples. Controland target samples can be diluted, if desired, prior to hybridization toequalize fluorescence intensities.

[0081] 6. Supports

[0082] Supports can be made of a variety of materials, such as glass,silica, plastic, nylon or nitrocellulose. Supports are preferably rigidand have a planar surface. Supports typically have from 1-10,000,000discrete spatially addressable regions, or cells. Supports having10-1,000,000 or 100-100,000 or 1000-100,000 cells are common. Thedensity of cells is typically at least 1000, 10,000, 100,000 or1,000,000 cells within a square centimeter. Typically a single probe percell. In some supports, all cells are occupied by pooled mixtures ofprobes. In other supports, some cells are occupied by pooled mixtures ofprobes, and other cells are occupied, at least to the degree of purityobtainable by synthesis methods, by a single type of polynucleotide. Thestrategies for probe design described in the present application can becombined with other strategies, such as those described by WO 95/11995,EP 717,113 and WO 97/29212 in the same array.

[0083] The location and sequence of each different polynucleotide probein the array is generally known. Moreover, the large number of differentprobes can occupy a relatively small area providing a high density arrayhaving a probe density of generally greater than about 60, moregenerally greater than about 100, and most generally greater than about600, often greater than about 1000, more often greater than about 5,000,most often greater than about 10,000, preferably greater than about40,000 more preferably greater than about 100,000, and most preferablygreater than about 400,000 different polynucleotide probes per cm². Thesmall surface area of the array (often less than about 10 cm²,preferably less than about 5 cm² more preferably less than about 2 cm²,and most preferably less than about 1.6 cm²) permits the use of smallsample volumes and extremely uniform hybridization conditions

[0084] 7. Synthesis of Probe Arrays

[0085] Arrays of probes can be synthesized in a step-by-step manner on asupport or can be attached in presynthesized form. A preferred method ofsynthesis is VLSIPS™ (see Fodor et al., 1991, Fodor et al., 1993, Nature364, 555-556; McGall et al., U.S. Ser. No. 08/445,332; U.S. Pat. No.5,143,854; EP 476,014), which entails the use of light to direct thesynthesis of polynucleotide probes in high-density, miniaturized arrays.Algorithms for design of masks to reduce the number of synthesis cyclesare described by Hubbel et al., U.S. Pat. No. 5,571,639 and U.S. Pat.No. 5,593,839. Arrays can also be synthesized in a combinatorial fashionby delivering monomers to cells of a support by mechanically constrainedflowpaths. See Winkler et al., EP 624,059. Arrays can also besynthesized by spotting monomers reagents on to a support using an inkjet printer. See id.; Pease et al., EP 728,520.

[0086] After hybridization of control and target samples to an arraycontaining one or more probe sets as described above and optionalwashing to remove unbound and nonspecifically bound probe, thehybridization intensity for the respective samples is determined foreach probe in the array. For fluorescent labels, hybridization intensitycan be determined by, for example, a scanning confocal microscope inphoton counting mode. Appropriate scanning devices are described bye.g., Trulson et al., U.S. Pat. No. 5,578,832; Stem et al., U.S. Pat.No. 5,631,734 and are available from Affymetrix, Inc., under theGeneChip™ label. Some types of label provide a signal that can beamplified by enzymatic methods (see Broude, et al., Proc. Natl. Acad.Sci. U.S.A. 91, 3072-3076 (1994))

[0087] 8. Design of Arrays

(A) Customized and Generic Arrays

[0088] The design of arrays for expression monitoring is generallydescribed, for example, WO 97/27317 and WO 97/10365 (these referencesare herein incorporated by reference). There are two principalcategories of arrays. One type of array detects the presence and/orlevels of particular mRNA sequences that are known in advance. In thesearrays, polynucleotide probes can be selected to hybridize to particularpreselected subsequences of mRNA gene sequence. Such expressionmonitoring arrays can include a plurality of probes for each mRNA to bedetected. For analysis of mRNA nucleic acids, the probes are designed tobe complementary to the region of the mRNA that is incorporated into thenucleic acids (i.e., the 3′ end). The array can also include one or morecontrol probes.

[0089] Generic arrays can include all possible nucleotides of a givenlength; that is, polynucleotides having sequences corresponding to everypermutation of a sequence. Thus since the polynucleotide probes of thisinvention preferably include up to 4 bases (A, G, C, T) or (A, G, C, U)or derivatives of these bases, an array having all possible nucleotidesof length X contains substantially 4^(X) different nucleic acids (e.g.,16 different nucleic acids for a 2 mer, 64 different nucleic acids for a3 mer, 65536 different nucleic acids for an 8 mer). Some small number ofsequences can be absent from a pool of all possible nucleotides of aparticular length due to synthesis problems, and inadvertent cleavage).An array comprising all possible nucleotides of length X refers to anarray having substantially all possible nucleotides of length X. Allpossible nucleotides of length X includes more than 90%, typically morethan 95%, preferably more than 98%, more preferably more than 99%, andmost preferably more than 99.9% of the possible number of differentnucleotides. Generic arrays are particularly useful for comparativehybridization analysis between two mRNA populations or nucleic acidsderived therefrom.

(B) Variations (1) Constant Regions

[0090] In both customized and generic array, probes can compriseadditional constant regions fused with the variable regions that mediatehybridization to target nucleic acid. In some arrays, constant regionsare double stranded thereby providing a site at which hybridized targetcan ligate to immobilized probes. A constant domain is a nucleotidesubsequence that is common to substantially all of the polynucleotideprobes. Constant domains are typically located at the terminus of thepolynucleotide probe closest to the substrate (i.e., attached to thelinker/anchor molecule). The constant regions can comprise virtually anysequence. Some constant regions comprise a sequence or subsequencecomplementary to the sense or antisense strand of a restriction site (anucleic acid sequence recognized by a restriction enzyme).

[0091] Constant regions can be synthesized de novo on the array orprepared in a separate procedure and then coupled intact to the array.Since the constant domain can be synthesized separately and then theintact constant subsequences coupled to the high density array, theconstant domain can be virtually any length. Some constant domains rangefrom 3 nucleotides to about 500 nucleotides in length, more typicallyfrom about 3 nucleotides in length to about 100 nucleotides in length,most typically from 3 nucleotides in length to about 50 nucleotides inlength. Constant domains can also range from 3 nucleotides to about 45nucleotides in length, or from 3 nucleotides in length to about 25nucleotides in length or from 3 to about 15 or even 10 nucleotides inlength. Constant domains can also range from about 5 nucleotides toabout 15 nucleotides in length.

(2) Control Probes

[0092] Either customized or generic probe arrays can contain controlprobes in addition to the probes described above.

(a) Normalization Controls

[0093] Normalization controls are typically perfectly complementary toone or more labeled reference polynucleotides that are added to thenucleic acid sample. The signals obtained from the normalizationcontrols after hybridization provide a control for variations inhybridization conditions, label intensity, reading and analyzingefficiency and other factors that can cause the signal of a perfecthybridization to vary between arrays. Signals (e.g., fluorescenceintensity) read from all other probes in the array can be divided by thesignal (erg., fluorescence intensity) from the control probes therebynormalizing the measurements.

[0094] Virtually any probe can serve as a normalization control.However, hybridization efficiency can vary with base composition andprobe length. Normalization probes can be selected to reflect theaverage length of the other probes present in the array, however, theycan also be selected to cover a range of lengths. The normalizationcontrol(s) can also be selected to reflect the (average) basecomposition of the other probes in the array. However one or a fewernormalization probes can be used and they can be selected such that theyhybridize well (i.e., no secondary structure) and do not match anytarget-specific probes.

[0095] Normalization probes can be localized at any position in thearray or at multiple positions throughout the array to control forspatial variation in hybridization efficiently. The normalizationcontrols can be located at the corners or edges of the array as well asin the middle of the array.

(b) Expression Level Controls

[0096] Expression level controls can be probes that hybridizespecifically with constitutively expressed genes in the biologicalsample. Expression level controls can be designed to control for theoverall health and metabolic activity of a cell. Examination of thecovariance of an expression level control with the expression level ofthe target nucleic acid can indicate whether measured changes orvariations in expression level of a gene is due to changes intranscription rate of that gene or to general variations in health ofthe cell. Thus, for example, when a cell is in poor health or lacking acritical metabolite the expression levels of both an active target geneand a constitutively expressed gene are expected to decrease. Theconverse can also be true. Thus where the expression levels of both anexpression level control and the target gene appear to both decrease orto both increase, the change can be attributed to changes in themetabolic activity of the cell as a whole, not to differentialexpression of the target gene in question. Conversely, where theexpression levels of the target gene and the expression level control donot covary, the variation in the expression level of the target gene canbe attributed to differences in regulation of that gene and not tooverall variations in the metabolic activity of the cell.

[0097] Virtually any constitutively expressed gene can provide asuitable target for expression level controls. Typically expressionlevel control probes can have sequences complementary to subsequences ofconstitutively expressed genes including, but not limited to the B-actingene, the transferrin receptor gene, the GAPDH gene, and the like.

(c) Mismatch Controls

[0098] Mismatch controls can also be provided for the probes to thetarget genes, for expression level controls or for normalizationcontrols. Mismatch controls are typically employed in customized arrayscontaining probes matched to known mRNA species. For example, some sucharrays contain a mismatch probe corresponding to each match probe. Themismatch probe is the same as its corresponding match probe except forat least one position of mismatch. A mismatched base is a base selectedso that it is not complementary to the corresponding base in the targetsequence to which the probe can otherwise specifically hybridize. One ormore mismatches are selected such that under appropriate hybridizationconditions (e.g. stringent conditions) the test or control probe can beexpected to hybridize with its target sequence, but the mismatch probecannot hybridize (or can hybridize to a significantly lesser extent).Mismatch probes can contain a central mismatch. Thus, for example, wherea probe is a 20 mer, a corresponding mismatch probe can have theidentical sequence except for a single base mismatch (e.g., substitutinga G, a C or a T for an A) at any of positions 6 through 14 (the centralmismatch).

[0099] In generic (e.g., random, arbitrary, or haphazard) arrays, sincethe target nucleic acid(s) are unknown perfect match and mismatch probescannot be a priori determined, designed, or selected. In this instance,the probes can be provided as pairs where each pair of probes differ inone or more preselected nucleotides. Thus, while it is not known apriori which of the probes in the pair is the perfect match, it is knownthat when one probe specifically hybridizes to a particular targetsequence, the other probe of the pair can act as a mismatch control forthat target sequence. The perfect match and mismatch probes need not beprovided as pairs, but can be provided as larger collections (e.g., 3,4, 5, or more) of probes that differ from each other in particularpreselected nucleotides.

[0100] In both customized and generic arrays mismatch probes can providea control for non-specific binding or cross-hybridization to a nucleicacid in the sample other than the target to which the probe iscomplementary. Mismatch probes thus can indicate whether a hybridizationis specific or not. For example, if the complementary target is presentthe perfect match probes can be consistently brighter than the mismatchprobes. In addition, if all central mismatches are present, the mismatchprobes can be used to detect a mutation. Finally, the difference inintensity between the perfect match and the mismatch probe (I(PM)-I(MM))can provide a good measure of the concentration of the hybridizedmaterial.

(d) Sample Preparation, Amplification, and Quantitation Controls

[0101] Arrays can also include sample preparation/amplification controlprobes. These can be probes that are complementary to subsequences ofcontrol genes selected because they do not normally occur in the nucleicacids of the particular biological sample being assayed. Suitable samplepreparation/amplification control probes can include, for example,probes to bacterial genes (e.g., Bio B) where the sample in question isa biological sample from a eukaryote.

[0102] The RNA sample can then be spiked with a known amount of thenucleic acid to which the sample preparation/amplification control probeis directed before processing. Quantification of the hybridization ofthe sample preparation/amplification control probe can then provide ameasure of alteration in the abundance of the nucleic acids caused byprocessing steps (e.g., PCR, reverse transcription, or in vitrotranscription).

[0103] Quantitation controls can be similar. Typically they can becombined with the sample nucleic acid(s) in known amounts prior tohybridization. They are useful to provide a quantitation reference andpermit determination of a standard curve for quantifying hybridizationamounts (concentrations).

[0104] 9. Methods of Detection

[0105] In one method of detection, mRNA or nucleic acid derivedtherefrom, typically in denatured form, are applied to an array. Thecomponent strands of the nucleic acids hybridize to complementaryprobes, which are identified by detecting label. Optionally, thehybridization signal of matched probes can be compared with that ofcorresponding mismatched or other control probes. Binding of mismatchedprobe serves as a measure of background and can be subtracted frombinding of matched probes. A significant difference in binding between aperfectly matched probes and a mismatched probes signifies that thenucleic acid to which the matched probes are complementary is present.Binding to the perfectly matched probes is typically at least 1.2, 1.5,2, 5 or 10 or 20 times higher than binding to the mismatched probes.

[0106] In a variation of the above method, nucleic acids are not labeledbut are detected by template-directed extension of a probe hybridized toa nucleic acid strand with the nucleic acid strand serving as atemplate. The probe is extended with a labeled nucleotide, and theposition of the label indicates, which probes in the array have beenextended. By performing multiple rounds of extension using differentbases bearing different labels, it is possible to determine the identityof additional bases in the tag than are determined throughcomplementarity with the probe to which the tag is hybridized. The useof target-dependent extension of probes is described by U.S. Pat. No.5,547,839.

[0107] In a further variation, probes can be extended with inosine. Theinosine strand can be labeled. The addition of degenerate bases, such asinosine (it can pair with all other bases), can increase duplexstability between the polynucleotide probe and the denatured singlestranded DNA nucleic acids. The addition of 1-6 inosines onto the end ofthe probes can increase the signal intensity in both hybridization andligation reactions on a generic ligation array. This can allow forligations at higher temperatures. The use of degenerate bases isdescribed in WO 97/27317.

[0108] Ligation reactions can offer improved discriminate between fullycomplementary hybrids and those that differ by one or more base pairs,particularly in cases where the mismatch is near the 5′ terminus of thepolynucleotide probes. Use of a ligation reaction in signal detectionincreases the stability of the hybrid duplex, improves hybridizationspecificity (particularly for shorter polynucleotide probes (e.g., 5 to12-mers), and optionally, provides additional sequence information.Ligation reactions used in signal detection are described in WO97/27317. Optionally, ligation reactions can be used in conjunction withtemplate-directed extension of probes, either by inosine or other bases.

[0109] 10. Analysis of Hybridization Patterns

[0110] The position of label is detected for each probe in the arrayusing a reader, such as described by U.S. Pat. No. 5,143,854, WO90/15070, and Trulson et al., supra. For customized arrays, thehybridization pattern can then be analyzed to determine the presenceand/or relative amounts or absolute amounts of known mRNA species insamples being analyzed as described in e.g., WO 97/10365. Comparison ofthe expression patterns of two samples is useful for identifying mRNAsand their corresponding genes that are differentially expressed betweenthe two samples.

[0111] The quantitative monitoring of expression levels for largenumbers of genes can prove valuable in elucidating gene function,exploring the causes and mechanisms of disease, and for the discovery ofpotential therapeutic and diagnostic targets. Expression monitoring canbe used to monitor the expression (transcription) levels of nucleicacids whose expression is altered in a disease state. For example, acancer can be characterized by the overexpression of a particular markersuch as the HER2 (c-erbB-2/neu) protooncogene in the case of breastcancer.

[0112] Expression monitoring can be used to monitor expression ofvarious genes in response to defined stimuli, such as a drug. This isespecially useful in drug research if the end point description is acomplex one, not simply asking if one particular gene is overexpressedor underexpressed. Therefore, where a disease state or the mode ofaction of a drug is not well characterized, the expression monitoringcan allow rapid determination of the particularly relevant genes.

[0113] In generic arrays, the hybridization pattern is also a measure ofthe presence and abundance of relative mRNAs in a sample, although it isnot immediately known, which probes correspond to which mRNAs in thesample.

[0114] However the lack of knowledge regarding the particular genes doesnot prevent identification of useful therapeutics. For example, if thehybridization pattern on a particular generic array for a healthy cellis known and significantly different from the pattern for a diseasedcell, then libraries of compounds can be screened for those that causethe pattern for a diseased cell to become like that for the healthycell. This provides a detailed measure of the cellular response to adrug.

[0115] Generic arrays can also provide a powerful tool for genediscovery and for elucidating mechanisms underlying complex cellularresponses to various stimuli. For example, generic arrays can be usedfor expression fingerprinting. Suppose it is found that the mRNA from acertain cell type displays a distinct overall hybridization pattern thatis different under different conditions (e.g., when harboring mutationsin particular genes, in a disease state). Then this pattern ofexpression (an expression fingerprint), if reproducible and clearlydifferentiable in the different cases can be used as a very detaileddiagnostic. It is not required that the pattern be fully interpretable,but just that it is specific for a particular cell state (and preferablyof diagnostic and/or prognostic relevance).

[0116] Both customized and generic arrays can be used in drug safetystudies. For example, if one is making a new antibiotic, then it shouldnot significantly affect the expression profile for mammalian cells. Thehybridization pattern can be used as a detailed measure of the effect ofa drug on cells, for example, as a toxicological screen.

[0117] The sequence information provided by the hybridization pattern ofa generic array can be used to identify genes encoding mRNAs hybridizedto an array. Such methods can be performed using DNA nucleic acids ofthe invention as the target nucleic acids described in WO 97/27317. DNAnucleic acids can be denatured and then hybridized to the complementaryregions of the probes, using standard conditions described in WO97/27317. The hybridization pattern indicates which probes arecomplementary to nucleic acid strands in the sample. Comparison of thehybridization pattern of two samples indicates which probes hybridize tonucleic acid strands that derive from mRNAs that are differentiallyexpressed between the two samples. These probes are of particularinterest, because they contain complementary sequence to mRNA speciessubject to differential expression. The sequence of such probes is knownand can be compared with sequences in databases to determine theidentity of the full-length mRNAs subject to differential expressionprovided that such mRNAs have previously been sequenced. Alternatively,the sequences of probes can be used to design hybridization probes orprimers for cloning the differentially expressed mRNAs. Thedifferentially expressed mRNAs are typically cloned from the sample inwhich the mRNA of interest was expressed at the highest level. In somemethods, database comparisons or cloning is facilitated by provision ofadditional sequence information beyond that inferable from probesequence by template dependent extension as described above.

[0118] 11. Screening for B Cell Activity Modulators

(A) Candidate Bioactive Agents

[0119] Having identified a number of suitable expression profiles, theinformation is used in a wide variety of ways. In a preferred method,the expression profiles can be used in conjunction with high throughputscreening techniques, to allow monitoring for expression profile genesafter treatment with a candidate agent, Zlokarnik, et al., Science 279,84-8 (1998), Heid et al., Genome Res. (1996) 6: 986. In a preferredmethod, the candidate agents are added to cells.

[0120] The term “candidate bioactive agent” or “drug candidate” orgrammatical equivalents as used herein describes any molecule, e.g.,protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, to be tested for bioactive agents that are capable ofdirectly or indirectly altering the activity of a B cell. In preferredmethods, the bioactive agents modulate the expression profiles, orexpression profile nucleic acids or proteins provided herein. In aparticularly preferred method, the candidate agents induce animmunosuppressive tolerant response, or maintain such a response asindicated, for example, by the effect of the agent on the expressionprofile, nucleic acids, proteins or B cell activity as further describedbelow. Generally a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a differential response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration or below the level ofdetection.

[0121] Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 100 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof. Particularly preferred are peptides.

[0122] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means. Knownpharmacological agents can be subjected to directed or random chemicalmodifications, such as acylation, alkylation, esterification,amidification to produce structural analogs.

[0123] In some preferred embodiment, the candidate bioactive agents areproteins. By “protein” herein is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein can be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains can be in either the (R) orthe (S) configuration. In some preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents can be used, for example toprevent or retard in vivo degradations.

[0124] In a preferred method, the candidate bioactive agents arenaturally occurring proteins or fragments of naturally occurringproteins. Thus, for example, cellular extracts containing proteins, orrandom or directed digests of proteinaceous cellular extracts, can beused. In this way libraries of procaryotic and eucaryotic proteins canbe made for screening in the methods of the invention. The libraries canbe bacterial, fungal, viral, and mammalian proteins, with the latterbeing preferred, and human proteins.

[0125] In some methods, the candidate bioactive agents are peptides offrom about 5 to about 30 amino acids, with from about 5 to about 20amino acids being preferred, and from about 7 to about 15 beingparticularly preferred. The peptides can be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. By “randomized” or grammatical equivalents herein ismeant that each nucleic acid and peptide consists of essentially randomnucleotides and amino acids, respectively. Since generally these randompeptides (or nucleic acids, discussed below) are chemically synthesized,they can incorporate any nucleotide or amino acid at any position. Thesynthetic process can be designed to generate randomized proteins ornucleic acids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

[0126] In some methods, the library can be fully randomized, with nosequence preferences or constants at any position. In other methods, thelibrary can be biased. Some positions within the sequence are eitherheld constant, or are selected from a limited number of possibilities.For example, in some methods, the nucleotides or amino acid residues arerandomized within a defined class, for example, of hydrophobic aminoacids, hydrophilic residues, sterically biased (either small or large)residues, towards the creation of nucleic acid binding domains, thecreation of cysteines, for cross-linking, prolines for SH-3 domains,serines, threonines, tyrosines or histidines for phosphorylation sites,or to purines. In other methods, the candidate bioactive agents arenucleic acids, as defined above.

[0127] As described above generally for proteins, nucleic acid candidatebioactive agents can be naturally occurring nucleic acids, randomnucleic acids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eucaryotic genomes can be used as is outlined above forproteins.

[0128] In some methods, the candidate bioactive agents are organicchemical moieties.

(B) Drug Screening Methods

[0129] Several different drug screening methods can be accomplished toidentify drugs or bioactive agents that modulate B cell activity. Onesuch method is the screening of candidate agents that can induce aparticular expression profile, thus preferably generating the associatedphenotype. Candidate agents that can mimic or produce an expressionprofile similar to an immunosuppressive expression profile as shownherein is expected to result in the immunosuppressive phenotype.Similarly, candidate agents that can mimic or produce an expressionprofile similar to a tolerant expression profile as shown herein isexpected to result in the tolerant phenotype. Thus, in some methods,candidate agents can be determined that mimic an expression profile orchange one profile to another.

[0130] In other methods, after having identified the differentiallyexpressed genes important in any one state, candidate agent screeningcan be run to alter the expression of individual genes. For example,particularly in the case of target genes whose presence or absence isunique between two states, screening for modulators of the target geneexpression can be done.

[0131] In other methods, screening can be done to alter the biologicalfunction of the expression product of the differentially expressed gene.Again, having identified the importance of a gene in a particular state,screening for agents that bind and/or modulate the biological activityof the gene product can be performed as outlined below.

[0132] Thus, screening of candidate agents that modulate B cell activityeither at the level of gene expression or protein level can beaccomplished.

[0133] In some methods, a candidate agent can be administered to B cellsin any state, that thus has an associated B cell activity expressionprofile. By “administration” or “contacting” herein is meant that thecandidate agent is added to the cells in such a manner as to allow theagent to act upon the cell, whether by uptake and intracellular action,or by action at the cell surface. In some embodiments, nucleic acidencoding a proteinaceous candidate agent (i.e., a peptide) can be putinto a viral construct such as a retroviral construct and added to thecell, such that expression of the peptide agent is accomplished; see PCTUS97/01019, hereby expressly incorporated by reference.

[0134] Once the candidate agent has been administered to the cells, thecells can be washed if desired and are allowed to incubate underpreferably physiological conditions for some period of time. The cellsare then harvested and a new gene expression profile is generated, asoutlined herein.

[0135] For example, activated B cells can be screened for agents thatproduce a tolerant phenotype. A change in at least one gene of theexpression profile indicates that the agent has an effect on B cellactivity. In a preferred method, an immunosuppressive tolerant profileis induced or maintained, before, during, and/or after stimulation withantigen. By defining such a signature for immunological tolerance,screens for new drugs that mimic the tolerance phenotype can be devised.With this approach, the drug target need not be known and need not berepresented in the original expression screening platform, nor does thelevel of transcript for the target protein need to change. In somemethods, the agent induces or maintains a profile which indicates aselective block immune response while still permitting tolerance to beactively (re)established. For example, in one such embodiment, the agentsuppresses at least one of Egr-1, Egr-2, cmyc and c-fos while sparingupregulation of NAB2.

[0136] In some preferred methods, screens can be done on individualgenes and gene products. After having identified a particulardifferentially expressed gene as important in a particular state,screening of modulators of either the expression of the gene or the geneproduct itself can be completed. The gene products of differentiallyexpressed genes are sometimes referred to herein as “B lymphocytemodulator proteins” or BLMPs.

[0137] Thus, in some preferred methods, screening for modulators ofexpression of specific genes can be completed. This will be done asoutlined above, but in general the expression of only one or a few genesare evaluated. In some methods, screens are designed to first findcandidate agents that can bind to differentially expressed proteins, andthen these agents can be used in other assays that evaluate the abilityof the candidate agent to modulate differentially expressed activity.There are a number of different assays which can be completed, such asbinding assays and activity assays.

[0138] In a preferred method, binding assays are performed. In general,purified or isolated gene product is used; that is, the gene products ofone or more differentially expressed nucleic acids are made. Using thenucleic acids of the present invention which encode a differentiallyexpressed protein in a B cell state, a variety of expression vectors canbe made. The expression vectors can be either self-replicatingextrachromosomal vectors or vectors which integrate into a host genome.Generally, these expression vectors include transcriptional andtranslational regulatory nucleic acid operably linked to the nucleicacid encoding a differentially expressed protein. The term “controlsequences” refers to DNA sequences necessary for the expression of anoperably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0139] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice. The transcriptionaland translational regulatory nucleic acid will generally be appropriateto the host cell used to express a differentially expressed protein; forexample, transcriptional and translational regulatory nucleic acidsequences from Bacillus are preferably used to express a differentiallyexpressed protein in Bacillus. Numerous types of appropriate expressionvectors, and suitable regulatory sequences are known in the art for avariety of host cells.

[0140] In general, the transcriptional and translational regulatorysequences can include, but are not limited to, promoter sequences,ribosomal binding sites, transcriptional start and stop sequences,translational start and stop sequences, and enhancer or activatorsequences. In a preferred method, the regulatory sequences include apromoter and transcriptional start and stop sequences.

[0141] Promoter sequences encode either constitutive or induciblepromoters. The promoters can be either naturally occurring promoters orhybrid promoters. Hybrid promoters, which combine elements of more thanone promoter, are also known in the art, and are useful in the presentinvention.

[0142] In addition, the expression vector can comprise additionalelements. For example, the expression vector can have two replicationsystems, thus allowing it to be maintained in two organisms, for examplein mammalian or insect cells for expression and in a procaryotic hostfor cloning and amplification. Furthermore, for integrating expressionvectors, the expression vector contains at least one sequence homologousto the host cell genome, and preferably two homologous sequences whichflank the expression construct. The integrating vector can be directedto a specific locus in the host cell by selecting the appropriatehomologous sequence for inclusion in the vector. Constructs forintegrating vectors are well known in the art. Preferred methods toeffect homologous recombination are described in PCT US93/03868 and PCTUS98/05223, hereby incorporated by reference.

[0143] In some methods, the expression vector contains a selectablemarker gene to allow the selection of transformed host cells. Selectiongenes are well known in the art and will vary with the host cell used.

[0144] A preferred expression vector system is a retroviral vectorsystem such as is generally described in PCT/US97/01019 andPCT/US97/01048, both of which are hereby expressly incorporated byreference.

[0145] The differentially expressed proteins of the present inventionare produced by culturing a host cell transformed with an expressionvector containing nucleic acid encoding a differentially expressedprotein, under the appropriate conditions to induce or cause expressionof a differentially expressed protein. The conditions appropriate fordifferentially expressed protein expression will vary with the choice ofthe expression vector and the host cell, and will be easily ascertainedby one skilled in the art through routine experimentation. For example,the use of constitutive promoters in the expression vector will requireoptimizing the growth and proliferation of the host cell, while the useof an inducible promoter requires the appropriate growth conditions forinduction. In some methods, the timing of the harvest is important. Forexample, the baculoviral systems used in insect cell expression arelytic viruses, and thus harvest time selection can be crucial forproduct yield.

[0146] Appropriate host cells include yeast, bacteria, archebacteria,fungi, and insect and animal cells, including mamnmalian cells. Ofparticular interest are Drosophila melangaster cells, Saccharomycescerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells. In somepreferred methods, B cells are host cells as provided herein, which forexample, include non-recombinant cell lines, such as primary cell lines.In addition, purified primary B cells derived from either transgenic ornon-transgenic strains can also be used. The B cells can be in aparticular state, or be induced to be in a particular state. The hostcell can alternatively be a B cell known to have a B cell disorder.

[0147] In a preferred method, the differentially expressed proteins areexpressed in mammalian cells. Mammalian expression systems can includeretroviral systems. A mammalian promoter is any DNA sequence capable ofbinding mammalian RNA polymerase and initiating the downstream (3′)transcription of a coding sequence for differentially expressed proteininto mRNA. A promoter will have a transcription initiating region, whichis usually placed proximal to the 5′ end of the coding sequence, and aTATA box, using a located 25-30 base pairs upstream of the transcriptioninitiation site. The TATA box is thought to direct RNA polymerase II tobegin RNA synthesis at the correct site. A mammalian promoter will alsocontain an upstream promoter element (enhancer element), typicallylocated within 100 to 200 base pairs upstream of the TATA box. Anupstream promoter element determines the rate at which transcription isinitiated and can act in either orientation. Of particular use asmammalian promoters are the promoters from mammalian viral genes, sincethe viral genes are often highly expressed and have a broad host range.Examples include the SV40 early promoter, mouse mammary tumor virus LTRpromoter, adenovirus major late promoter, herpes simplex virus promoter,and the CMV promoter.

[0148] Typically, transcription termination and polyadenylationsequences recognized by mammalian cells are regulatory regions located3′ to the translation stop codon and thus, together with the promoterelements, flank the coding sequence. The 3′ terminus of the mature mRNAis formed by site-specific post-translational cleavage andpolyadenylation. Examples of transcription terminator and polyadenlytionsignals include those derived form SV40.

[0149] The methods of introducing nucleic acid into mammalian hosts, aswell as other hosts, is well known in the art, and will vary with thehost cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, viral infection, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

[0150] In some methods, differentially expressed proteins are expressedin bacterial systems which are well known in the art.

[0151] In other methods, differentially expressed proteins can beproduced in insect cells. Expression vectors for the transformation ofinsect cells, and in particular, baculovirus-based expression vectors,are well known in the art.

[0152] In some methods, differentially expressed proteins are producedin yeast cells. Yeast expression systems are well known in the art, andinclude expression vectors for Saccharomyces cerevisiae, Candidaalbicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilisand K. lactis, Pichia guillerimondii and P. pastoris,Schizosaccharomyces pombe, and Yarrowia lipolytica.

[0153] A differentially expressed protein can also be made as a fusionprotein, using techniques well known in the art. For example, for thecreation of monoclonal antibodies, if the desired epitope is small, thedifferentially expressed protein can be fused to a carrier protein toform an immunogen. Alternatively, a differentially expressed protein canbe made as a fusion protein to increase expression. For example, when adifferentially expressed protein is a differentially expressed peptide,the nucleic acid encoding the peptide can be linked to other nucleicacid for expression purposes. Similarly, differentially expressedproteins of the invention an be linked to protein labels, such as greenfluorescent protein (GFP), red fluorescent protein (RFP), yellowfluorescent protein (YFP), and blue fluorescent protein (BFP).

[0154] Preferably, the proteins are recombinant. A “recombinant protein”is a protein made using recombinant techniques, i.e., through theexpression of a recombinant nucleic acid as depicted above. Arecombinant protein is distinguished from naturally occurring protein byat least one or more characteristics. For example, the protein can beisolated or purified away from some or all of the proteins and compoundswith which it is normally associated in its wild type host, and thus canbe substantially pure. For example, an isolated protein is unaccompaniedby at least some of the material with which it is normally associated inits natural state, preferably constituting at least about 0.5%, morepreferably at least about 5% by weight of the total protein in a givensample. A substantially pure protein comprises at least about 75% byweight of the total protein, with at least about 80% being preferred,and at least about 90% being particularly preferred. The definitionincludes the production of a differentially expressed protein from oneorganism in a different organism or host cell. Alternatively, theprotein can be made at a significantly higher concentration than isnormally seen, through the use of a inducible promoter or highexpression promoter, such that the protein is made at increasedconcentration levels. Alternatively, the protein can be in a form notnormally found in nature, as in the addition of an epitope tag or aminoacid substitutions, insertions and deletions, as discussed below.

[0155] In some preferred methods, when the differentially expressedprotein is to be used to generate antibodies, the protein must share atleast one epitope or determinant with the full length transcriptionproduct of the differentially expressed nucleic acids shown herein. By“epitope” or “determinant” herein is meant a portion of a protein whichwill generate and/or bind an antibody. Thus, in most instances,antibodies made to a smaller protein should be able to bind to the fulllength protein. In a preferred embodiment, the epitope is unique; thatis, antibodies generated to a unique epitope show little or nocross-reactivity.

[0156] In some preferred methods, the antibodies provided herein can becapable of reducing or eliminating the biological flnction of adifferentially expressed protein, as is described below. The addition ofantibodies (either polyclonal or preferably monoclonal) to the protein(or cells containing the differentially expressed protein) can reduce oreliminate the protein's activity. Generally, at least a 25% decrease inactivity is preferred, with at least about 50% being particularlypreferred and about a 95-100% decrease being especially preferred.

[0157] In addition, the proteins can be variant proteins, comprising onemore amino acid substitutions, insertions and deletions.

[0158] In a preferred method, a differentially expressed protein ispurified or isolated after expression. Differentially expressed proteinscan be isolated or purified in a variety of ways. Standard purificationmethods include electrophoretic, molecular, immunological andchromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography, and chromatofocusing.For example, a differentially expressed protein can be purified using astandard anti-differentially expressed protein antibody column.Ultrafiltration and diafiltration techniques, in conjunction withprotein concentration, are also useful. For general guidance in suitablepurification techniques, see Scopes, R., Protein Purification,Springer-Verlag, NY (1982). The degree of purification necessary willvary depending on the use of the differentially expressed protein. Insome instances no purification will be necessary.

[0159] Once the gene product of the differentially expressed gene ismade, binding assays can be done. These methods comprise combining adifferentially expressed protein and a candidate bioactive agent, anddetermining the binding of the candidate agent to the differentiallyexpressed protein. Preferred methods utilize a human differentiallyexpressed protein, although other mammalian proteins can also be used,including rodents (mice, rats, hamsters, guinea pigs), farm animals(cows, sheep, pigs, horses) and primates. These latter methods can bepreferred for the development of animal models of human disease. In somemethods, variant or derivative differentially expressed proteins can beused, including deletion differentially expressed proteins as outlinedabove.

[0160] The assays herein utilize differentially expressed proteins asdefined herein. In some assays, portions of differentially expressedproteins can be utilized. In other assays, portions havingdifferentially expressed activity can be used. In addition, the assaysdescribed herein can utilize either isolated differentially expressedproteins or cells comprising the differentially expressed proteins. Insome methods, the differentially expressed protein or the candidateagent is non-difflisably bound to an insoluble support having isolatedsample receiving areas (e.g., a microtiter plate or an array). Theinsoluble supports can be made of any composition to which thecompositions can be bound, is readily separated from soluble material,and is otherwise compatible with the overall method of screening. Thesurface of such supports can be solid or porous and of any convenientshape. Examples of suitable insoluble supports include microtiterplates, arrays, membranes and beads. These are typically made of glass,plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose,and teflon™. Microtiter plates and arrays are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples. In some cases magneticbeads and the like are included. The particular manner of binding of thecomposition is not crucial so long as it is compatible with the reagentsand overall methods of the invention, maintains the activity of thecomposition and is nondiffusable. Preferred methods of binding includethe use of antibodies (which do not sterically block either the ligandbinding site or activation sequence when the protein is bound to thesupport), direct binding to “sticky” or ionic supports, chemicalcrosslinking, the synthesis of the protein or agent on the surface.Following binding of the protein or agent, excess unbound material isremoved by washing. The sample receiving areas can then be blockedthrough incubation with bovine serum albumin (BSA), casein or otherinnocuous protein or other moiety. Also included in this invention arescreening assays wherein solid supports are not used.

[0161] In other methods, the differentially expressed protein is boundto the support, and a candidate bioactive agent is added to the assay.Alternatively, the candidate agent is bound to the support and thedifferentially expressed protein is added. Novel binding agents includespecific antibodies, non-natural binding agents identified in screens ofchemical libraries, and peptide analogs. Of particular interest arescreening assays for agents that have a low toxicity for human cells. Awide variety of assays can be used for this purpose, including labeledin vitro protein-protein binding assays, electrophoretic mobility shiftassays, immunoassays for protein binding, functional assays (such asphosphorylation assays) and the like.

[0162] The determination of the binding of the candidate bioactive agentto a differentially expressed protein can be done in a number of ways.In some methods, the candidate bioactive agent is labeled, and bindingdetermined directly. For example, this can be done by attaching all or aportion of a differentially expressed protein to a solid support, addinga labeled candidate agent (for example a fluorescent label), washing offexcess reagent, and determining whether the label is present on thesolid support. Various blocking and washing steps can be utilized.

[0163] By “labeled” herein is meant that the compound is either directlyor indirectly labeled with a label which provides a detectable signal,e.g., radioisotope, fluorescers, enzyme, antibodies, particles such asmagnetic particles, chemiluminescers, or specific binding molecules.Specific binding molecules include pairs, such as biotin andstreptavidin, digoxin and antidigoxin. For the specific binding members,the complementary member would normally be labeled with a molecule whichprovides for detection, in accordance with known procedures, as outlinedabove. The label can directly or indirectly provide a detectable signal.

[0164] In some methods, only one of the components is labeled. Forexample, the proteins (or proteinaceous candidate agents) can be labeledat tyrosine positions using ¹²⁵I, or with fluorophores. Alternatively,more than one component can be labeled with different labels; using ¹²⁵Ifor the proteins, for example, and a fluorophor for the candidateagents.

[0165] In other methods, the binding of the candidate bioactive agent isdetermined through the use of competitive binding assays. In thismethod, the competitor is a binding moiety known to bind to the targetmolecule such as an antibody, peptide, binding partner, or ligand. Undercertain circumstances, there can be competitive binding as between thebioactive agent and the binding moiety, with the binding moietydisplacing the bioactive agent. This assay can be used to determinecandidate agents which interfere with binding between differentiallyexpressed proteins and the competitor.

[0166] In some methods, the candidate bioactive agent is labeled. Eitherthe candidate bioactive agent, or the competitor, or both, is addedfirst to the protein for a time sufficient to allow binding, if present.Incubations can be performed at any temperature which facilitatesoptimal activity, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but can also be optimized to facilitaterapid high through put screening. Typically between 0.1 and 1 hour willbe sufficient. Excess reagent is generally removed or washed away. Thesecond component is then added, and the presence or absence of thelabeled component is followed, to indicate binding.

[0167] In other methods, the competitor is added first, followed by thecandidate bioactive agent. Displacement of the competitor is anindication that the candidate bioacfive agent is binding to thedifferentially expressed protein and thus is capable of binding to, andpotentially modulating, the activity of the differentially expressedprotein. In this method, either component can be labeled. For example,if the competitor is labeled, the presence of label in the wash solutionindicates displacement by the agent. Alternatively, if the candidatebioactive agent is labeled, the presence of the label on the supportindicates displacement.

[0168] In other methods, the candidate bioactive agent is added first,with incubation and washing, followed by the competitor. The absence ofbinding by the competitor can indicate that the bioactive agent is boundto the differentially expressed protein with a higher affinity. Thus, ifthe candidate bioactive agent is labeled, the presence of the label onthe support, coupled with a lack of competitor binding, can indicatethat the candidate agent is capable of binding to the differentiallyexpressed protein.

[0169] Competitive binding methods can also be run as differentialscreens. These methods can comprise a differentially expressed proteinand a competitor in a first sample. A second sample comprises acandidate bioactive agent, a differentially expressed protein and acompetitor. The binding of the competitor is determined for bothsamples, and a change, or difference in binding between the two samplesindicates the presence of an agent capable of binding to thedifferentially expressed protein and potentially modulating itsactivity. If the binding of the competitor is different in the secondsample relative to the first sample, the agent is capable of binding tothe differentially expressed protein.

[0170] Other methods utilize differential screening to identify drugcandidates that bind to the native differentially expressed protein, butcannot bind to modified differentially expressed proteins. The structureof the differentially expressed protein can be modeled, and used inrational drug design to synthesize agents that interact with that site.Drug candidates that affect differentially expressed bioactivity arealso identified by screening drugs for the ability to either enhance orreduce the activity of the protein.

[0171] In some methods, screening for agents that modulate the activityof differentially expressed proteins are performed. In general, thiswill be done on the basis of the known biological activity of thedifferentially expressed protein. In these methods, a candidatebioactive agent is added to a sample of the differentially expressedprotein, as above, and an alteration in the biological activity of theprotein is determined. “Modulating the activity” includes an increase inactivity, a decrease in activity, or a change in the type or kind ofactivity present. Thus, in these methods, the candidate agent shouldboth bind to differentially expressed (although this may not benecessary), and alter its biological or biochemical activity as definedherein. The methods include both in vitro screening methods, as aregenerally outlined above, and in vivo screening of cells for alterationsin the presence, distribution, activity or amount of the differentiallyexpressed protein.

[0172] Some methods comprise combining a differentially expressed sampleand a candidate bioactive agent, then evaluating the effect on B cellactivity. By “differentially expressed activity” or grammaticalequivalents herein is meant one of B cell biological activities,including, but not limited to, its ability to affect suppression,tolerance and activation. One activity herein is the capability to bindto a target gene, or modulate an expression profile. Preferably,expression profiles are induced or maintained and/or the desired B cellstate is induced or maintained.

[0173] In other methods, the activity of the differentially expressedprotein is increased; in other methods, the activity of thedifferentially expressed protein is decreased. Thus, bioactive agentsthat are antagonists are preferred in some methods, and bioactive agentsthat are agonists can be preferred in other methods.

[0174] The invention provides methods for screening for bioactive agentscapable of modulating the activity of a differentially expressedprotein. These methods comprise adding a candidate bioactive agent, asdefined above, to a cell comprising differentially expressed proteins.Preferred cell types include almost any cell. The cells contain arecombinant nucleic acid that encodes a differentially expressedprotein. In a preferred method, a library of candidate agents are testedon a plurality of cells. The effect of the candidate agent on B cellactivity is then evaluated.

[0175] Positive controls and negative controls can be used in theassays. Preferably all control and test samples are performed in atleast triplicate to obtain statistically significant results. Incubationof all samples is for a time sufficient for the binding of the agent tothe protein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples can be counted in a scintillation counter to determine theamount of bound compound.

[0176] A variety of other reagents can be included in the screeningassays. These include reagents like salts, neutral proteins (e.g.,albumin and detergents) which can be used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Reagents that otherwise improve the efficiency of theassay, (such as protease inhibitors, nuclease inhibitors, anti-microbialagents) can also be used. The mixture of components can be added in anyorder that provides for the requisite binding.

[0177] The components provided herein for the assays provided herein canalso be combined to form kits. The kits can be based on the use of theprotein and/or the nucleic acid encoding the differentially expressedproteins. Assays regarding the use of nucleic acids are furtherdescribed below.

(C) Animal Models

[0178] In a preferred method, nucleic acids which encode differentiallyexpressed proteins or their modified forms can also be used to generateeither transgenic animals, including “knock-in” and “knock out” animalswhich, in turn, are useful in the development and screening oftherapeutically useful reagents. A non-human transgenic animal (e.g., amouse or rat) is an animal having cells that contain a transgene, whichtransgene is introduced into the animal or an ancestor of the animal ata prenatal, e.g., an embryonic stage. A transgene is a DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops, and can include both the addition of all or part of a gene orthe deletion of all or part of a gene. In some methods, cDNA encoding adifferentially expressed protein can be used to clone genomic DNAencoding a differentially expressed protein in accordance withestablished techniques and the genomic sequences used to generatetransgenic animals that contain cells which either express (oroverexpress) or suppress the desired DNA. Methods for generatingtransgenic animals, particularly animals such as mice or rats, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells would betargeted for a differentially expressed protein transgene incorporationwith tissue-specific enhancers. Transgenic animals that include a copyof a transgene encoding a differentially expressed protein introducedinto the germ line of the animal at an embryonic stage can be used toexamine the effect of increased expression of the desired nucleic acid.Such animals can be used as tester animals for reagents thought toconfer protection from, for example, pathological conditions associatedwith its overexpression. In accordance with this facet of the invention,an animal is treated with the reagent and a reduced incidence of thepathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition. Similarly, non-human homologues of adifferentially expressed protein can be used to construct a transgenicanimal comprising a differentially expressed protein “knock out” animalwhich has a defective or altered gene encoding a differentiallyexpressed protein as a result of homologous recombination between theendogenous gene encoding a differentially expressed protein and alteredgenomic DNA encoding a differentially expressed protein introduced intoan embryonic cell of the animal. For example, cDNA encoding adifferentially expressed protein can be used to clone genomic DNAencoding a differentially expressed protein in accordance withestablished techniques. A portion of the genomic DNA encoding adifferentially expressed protein can be deleted or replaced with anothergene, such as a gene encoding a selectable marker which can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell (1987) 51: 503 for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell (1992) 69: 915). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of a differentially expressed proteinpolypeptide.

[0179] Animal models for B cell related disorders, or having aparticular state of B cell activity can include, for example, geneticmodels. For example, such animal models can include the nonobesediabetic (NOD) mouse (see, e.g., McDuffie, M., Curr Opin Immunol. (1998)10(6):704-9; Tochino, Y., Crit Rev Immunol (1987) 8(1): 49-81), andexperimental autoimmune encephalomyelitis (EAE) (see, e.g., Wong, F. S.,Immunol Rev (1999) 169: 93-104). See also Schwartz, R. S. and Datta, S.K., Autoimmunity and Autoimmune Diseases, Ch. 31, in FundamentalImmunology, Paul, W. E. (ed.) (Raven Press 1989). Other models caninclude studies involving transplant rejection.

[0180] Animal models exhibiting B cell related disorder-like symptomscan be engineered by utilizing, for example, differentially expressedsequences in conjunction with techniques for producing transgenicanimals that are well known to those of skill in the art. For example,gene sequences can be introduced into, and overexpressed in, the genomeof the animal of interest, or, if endogenous target gene sequences arepresent, they can either be overexpressed or, alternatively, can bedisrupted in order to underexpress or inactivate target gene expression.

[0181] In order to overexpress a target gene sequence, the codingportion of the target gene sequence can be ligated to a regulatorysequence which is capable of driving gene expression in the animal andcell type of interest. Such regulatory regions will be well known tothose of skill in the art, and can be utilized in the absence of undueexperimentation.

[0182] For underexpression of an endogenous target gene sequence, such asequence can be isolated and engineered such that when reintroduced intothe genome of the animal of interest, the endogenous target gene alleleswill be inactivated. Preferably, the engineered target gene sequence isintroduced via gene targeting such that the endogenous target sequenceis disrupted upon integration of the engineered target sequence into theanimal's genome.

[0183] Animals of any species, including, but not limited to, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees can be used togenerate animal models of B cell related disorders or being aperpetually desired state of the B cell.

(D) Nucleic Acid Based Therapeutics

[0184] Nucleic acids encoding differentially expressed polypeptides,antagonists or agonists can also be used in gene therapy. Broadlyspeaking, a gene therapy vector is an exogenous polynucleotide whichproduces a medically useful phenotypic effect upon the mammalian cell(s)into which it is transferred. A vector can or can not have an origin ofreplication. For example, it is useful to include an origin ofreplication in a vector for propagation of the vector prior toadministration to a patient. However, the origin of replication canoften be removed before administration if the vector is designed tointegrate into host chromosomal DNA or bind to host mRNA or DNA. Vectorsused in gene therapy can be viral or nonviral. Viral vectors are usuallyintroduced into a patient as components of a virus. Nonviral vectors,typically dsDNA, can be transferred as naked DNA or associated with atransfer-enhancing vehicle, such as a receptor-recognition protein,lipoamine, or cationic lipid.

(1) Viral-Based Methods

[0185] Viral vectors, such as retroviruses, adenoviruses,adenoassociated viruses and herpes viruses, are often made up of twocomponents, a modified viral genome and a coat structure surrounding it(see generally Smith et al., Ann. Rev. Microbiol. (1995) 49, 807-838;this reference and all references cited therein are incorporated hereinby reference), although sometimes viral vectors are introduced in nakedform or coated with proteins other than viral proteins. Most currentvectors have coat structures similar to a wildtype virus. This structurepackages and protects the viral nucleic acid and provides the means tobind and enter target cells. However, the viral nucleic acid in a vectordesigned for gene therapy is changed in many ways. The goals of thesechanges are to disable growth of the virus in target cells whilemaintaining its ability to grow in vector form in available packaging orhelper cells, to provide space within the viral genome for insertion ofexogenous DNA sequences, and to incorporate new sequences that encodeand enable appropriate expression of the gene of interest. Thus, vectornucleic acids generally comprise two components: essential cis-actingviral sequences for replication and packaging in a helper line and thetranscription unit for the exogenous gene. Other viral functions areexpressed in trans in a specific packaging or helper cell line.

(a) Retroviruses

[0186] Retroviruses comprise a large class of enveloped viruses thatcontain single—stranded RNA as the viral genome. During the normal virallife cycle, viral RNA is reverse-transcribed to yield double-strandedDNA that integrates into the host genome and is expressed over extendedperiods. As a result, infected cells shed virus continuously withoutapparent harm to the host cell. The viral genome is small (approximately10 kb), and its prototypical organization is extremely simple,comprising three genes encoding gag, the group specific antigens or coreproteins; pol, the reverse transcriptase; and env, the viral envelopeprotein. The termini of the RNA genome are called long terminal repeats(LTRs) and include promoter and enhancer activities and sequencesinvolved in integration. The genome also includes a sequence requiredfor packaging viral RNA and splice acceptor and donor sites forgeneration of the separate envelope mRNA. Most retroviruses canintegrate only into replicating cells, although human immunodeficiencyvirus (HIV) appears to be an exception. This property restricts the useof retroviruses as vectors for gene therapy.

[0187] Retrovirus vectors are relatively simple, containing the 5′ and3′ LTRs, a packaging sequence, and a transcription unit composed of thegene or genes of interest, which is typically an expression cassette. Togrow such a vector, one must provide the missing viral functions intrans using a so-called packaging cell line. Such a cell is engineeredto contain integrated copies of gag, pol, and env but to lack apackaging signal so that no helper virus sequences become encapsidated.Additional features added to or removed from the vector and packagingcell line reflect attempts to render the vectors more efficacious orreduce the possibility of contamination by helper virus.

[0188] The main advantage of retroviral vectors is that they integrateand are therefore potentially capable of long-term expression. They canbe grown in relatively large amounts, but care is needed to ensure theabsence of helper virus.

(b) Adenoviruses

[0189] Adenoviruses comprise a large class of nonenveloped virusescontaining linear double-stranded DNA. The normal life cycle of thevirus does not require dividing cells and involves productive infectionin permissive cells during which large amounts of virus accumulate. Theproductive infection cycle takes about 32-36 hours in cell culture andcomprises two phases, the early phase, prior to viral DNA synthesis, andthe late phase, during which structural proteins and viral DNA aresynthesized and assembled into virions. In general, adenovirusinfections are associated with mild disease in humans.

[0190] Adenovirus vectors are somewhat larger and more complex thanretrovirus or AAV vectors, partly because only a small fraction of theviral genome is removed from most current vectors. If additional genesare removed, they are provided in trans to produce the vector, which sofar has proved difficult. Instead, two general types of adenovirus-basedvectors have been studied, E3-deletion and E1-deletion vectors. Someviruses in laboratory stocks of wildtype lack the E3 region and can growin the absence of helper. This ability does not mean that the E3 geneproducts are not necessary in the wild, only that replication incultured cells does not require them. Deletion of the E3 region allowsinsertion of exogenous DNA sequences to yield vectors capable ofproductive infection and the transient synthesis of relatively largeamounts of encoded protein.

[0191] Deletion of the E1 region disables the adenovirus, but suchvectors can still be grown because there exists an established humancell line (called “293”) that contains the El region of Ad5 and thatconstitutively expresses the El proteins. Most recent gene therapyapplications involving adenovirus have utilized E1 replacement vectorsgrown in 293 cells.

[0192] The main advantages of adenovirus vectors are that they arecapable of efficient episomal gene transfer in a wide range of cells andtissues and that they are easy to grow in large amounts. The maindisadvantage is that the host response to the virus appears to limit theduration of expression and the ability to repeat dosing, at least withhigh doses of first-generation vectors.

(c) Adeno-Associated Virus (AAV)

[0193] AAV is a small, simple, nonautonomous virus containing linearsinglestranded DNA. See Muzycka, Current Topics Microbiol. Immunol.(1992) 158, 97-129; this reference and all references cited therein areincorporated herein by reference. The virus requires co-infection withadenovirus or certain other viruses in order to replicate. AAV iswidespread in the human population, as evidenced by antibodies to thevirus, but it is not associated with any known disease. AAV genomeorganization is straightforward, comprising only two genes: rep and cap.The termini of the genome comprises terminal repeats (ITR) sequences ofabout 145 nucleotides.

[0194] AAV-based vectors typically contain only the ITR sequencesflanking the transcription unit of interest. The length of the vectorDNA cannot greatly exceed the viral genome length of 4680 nucleotides.Currently, growth of AAV vectors is cumbersome and involves introducinginto the host cell not only the vector itself but also a plasmidencoding rep and cap to provide helper functions. The helper plasmidlacks ITRs and consequently cannot replicate and package. In addition,helper virus such as adenovirus is often required. The potentialadvantage of AAV vectors is that they appear capable of long-termexpression in nondividing cells, possibly, though not necessarily,because the viral DNA integrates. The vectors are structurally simple,and they can therefore provoke less of a host-cell response thanadenovirus. A major limitation at present is that AAV vectors areextremely difficult to grow in large amounts.

(2) Non-Viral Gene Transfer Methods

[0195] Nonviral nucleic acid vectors used in gene therapy includeplasmids, RNAs, antisense oligonucleotides (e.g., methylphosphonate orphosphorothiolate), polyamide nucleic acids, and yeast artificialchromosomes (YACs). Such vectors typically include an expressioncassette for expressing a protein or RNA. The promoter in such anexpression cassette can be constitutive, cell type-specific,stage-specific, and/or modulatable (e.g., by hormones such asglucocorticoids; MMTV promoter). Transcription can be increased byinserting an enhancer sequence into the vector. Enhancers are cis-actingsequences of between 10 to 300bp that increase transcription by apromoter. Enhancers can effectively increase transcription when either5′ or 3′ to the transcription unit. They are also effective if locatedwithin an intron or within the coding sequence itself. Typically, viralenhancers are used, including SV40 enhancers, cytomegalovirus enhancers,polyoma enhancers, and adenovirus enhancers. Enhancer sequences frommammalian systems are also commonly used, such as the mouseimmunoglobulin heavy chain enhancer.

[0196] Gene therapy vectors of all kinds can also include a selectablemarker gene. Examples of suitable markers include, the dihydrofolatereductase gene (DHFR), the thymidine kinase gene (TK), or prokaryoticgenes conferring drug resistance, gpt (xanthine-guaninephosphoribosyltransferase, which can be selected for with mycophenolicacid; neo (neomycin phosphotransferase), which can be selected for withG418, hygromycin, or puromycin; and DHFR (dihydrofolate reductase),which can be selected for with methotrexate (Mulligan & Berg, Proc.Nati. Acad. Sci. U.S.A. (1981) 78, 2072; Southern & Berg, J. Mol. Appl.Genet. (1982) 1, 327).

[0197] Before integration, the vector has to cross many barriers whichcan result in only a very minor fraction of the DNA ever beingexpressed. Limitations to high level gene expression include: loss ofvector due to nucleases present in blood and tissues; inefficient entryof DNA into a cell; inefficient entry of DNA into the nucleus of thecell and preference of DNA for other compartments; lack of DNA stabilityin the nucleus (factor limiting nuclear stability can differ from thoseaffecting other cellular and extracellular compartments), efficiency ofintegration into the chromosome; and site of integration.

[0198] These potential losses of efficiency can be addressed byincluding additional sequences in a nonviral vector besides theexpression cassette from which the product effecting therapy is to beexpressed. The additional sequences can have roles in conferringstability both outside and within a cell, mediating entry into a cell,mediating entry into the nucleus of a cell and mediating integrationwithin nuclear DNA. For example, aptamer-like DNA structures, or otherprotein binding sites can be used to mediate binding of a vector to cellsurface receptors or to serum proteins that bind to a receptor therebyincreasing the efficiency of DNA transfer into the cell.

[0199] Other DNA sequences can directly or indirectly result inavoidance of certain compartments and preference for other compartments,from which escape or entry into the nucleus is more efficient. Other DNAsites and structures directly or indirectly bind to receptors in thenuclear membrane or to other proteins that go into the nucleus, therebyfacilitating nuclear uptake of a vector. Other DNA sequences directly orindirectly affect the efficiency of integration. For integration byhomologous recombination, important factors are the degree and length ofhomology to chromosomal sequences, as well as the frequency of suchsequences in the genome (e.g., alu repeats). The specific sequencemediating homologous recombination is also important, since integrationoccurs much more easily in transcriptionally active DNA. Methods andmaterials for constructing homologous targeting constructs are describedby e.g., Mansour et al., Nature (1988) 336: 348; Bradley et al.,Bio/Technology (1992) 10: 534.

[0200] For nonhomologous, illegitimate and site-specific recombination,recombination is mediated by specific sites on the therapy vector whichinteract with cell encoded recombination proteins (e.g., cre/lox andflp/frt systems). For example Baubonis & Sauer, Nuc. Acids Res. (1993)21, 2025-2029 report that a vector including a loxP site becomesintegrated at a loxP site in chromosomal DNA in the presence of creenzyme.

[0201] Nonviral vectors encoding products useful in gene therapy can beintroduced into an animal by means such as lipofection, biolistics,virosomes, liposomes, immunoliposomes, polycation: nucleic acidconjugates, naked DNA, artificial virions, agent-enhanced uptake of DNA,ex vivo transduction. Lipofection is described in e.g., U.S. Pat. Nos.5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are soldcommercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutrallipids that are suitable for efficient receptor-recognition lipofectionof polynucleotides include those of Felgner, WO 91/17424, WO 91/16024.

[0202] Unlike existing viral-based gene therapy vectors which can onlyincorporate a relatively small non-viral polynucleotide sequence intothe viral genome because of size limitations for packaging virionparticles, naked DNA or lipofection complexes can be used to transferlarge (e.g., 50-5,000 kb) exogenous polynucleotides into cells. Thisproperty of nonviral vectors is particularly advantageous since manygenes which can be delivered by therapy span over 100 kilobases (e.g.,amyloid precursor protein (APP) gene, Huntington's chorea gene) andlarge homologous targeting constructs or transgenes can be required forefficient integration. Optionally, such large genes can be delivered totarget cells as two or more fragments and reconstructed by homologousrecombination within a cell (see WO 92/03917).

(3) Applications of Gene Therapy

[0203] Gene therapy vectors can be delivered in vivo by administrationto an individual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application. Alternatively, vectors can bedelivered to cells ex vivo, such as cells explanted from an individualpatient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) oruniversal donor hematopoietic stem cells, followed by reimplantation ofthe cells into a patient, usually after selection for cells which haveincorporated the vector.

[0204] 12. Diagnostic Methods

[0205] In addition to assays, the creation of animal models, and nucleicacid based therepeutics, identification of important differentiallyexpressed genes allows the use of these genes in diagnosis (e.g.,diagnosis of cell states and abnormal B cell conditions). Disordersbased on mutant or variant differentially expressed genes can bedetermined. The invention also provides methods for identifying cellscontaining variant differentially expressed genes comprising determiningall or part of the sequence of at least one endogeneous differentiallyexpressed genes in a cell. As will be appreciated by those in the art,this can be done using any number of sequencing techniques. Theinvention also provides methods of identifying the differentiallyexpressed genotype of an individual comprising determining all or partof the sequence of at least one differentially expressed gene of theindividual. This is generally done in at least one tissue of theindividual, and can include the evaluation of a number of tissues ordifferent samples of the same tissue. The method can include comparingthe sequence of the sequenced differentially expressed gene to a knowndifferentially expressed gene, i.e., a wild-type gene.

[0206] The sequence of all or part of the differentially expressed genecan then be compared to the sequence of a known differentially expressedgene to determine if any differences exist. This can be done using anynumber of known sequence identity programs, such as Bestfit, and othersoutlined herein. In some preferred methods, the presence of a differencein the sequence between the differentially expressed gene of the patientand the known differentially expressed gene is indicative of a diseasestate or a propensity for a disease state, as outlined herein.

[0207] Similarly, diagnosis of B cell states can be done using themethods of the invention. By evaluating the gene expression profile of Bcells from a patient, the B cell state can be determined. This isparticularly useful to verify the action of a drug, for example animmunosuppressive drug. Other methods comprise administering the drug toa patient and removing a cell sample, particularly of B cells, from thepatient. The gene expression profile of the cell is then evaluated, asoutlined herein, for example by comparing it to the expression profilefrom an equivalent sample from a healthy individual. In this manner,both the efficacy (i.e., whether the correct expression profile is beinggenerated from the drug) and the dose (is the dosage correct to resultin the correct expression profile) can be verified.

[0208] The present discovery relating to the role of differentiallyexpressed in B cells thus provides methods for inducing or maintainingdiffering B cell states. In a preferred method, the differentiallyexpressed proteins, and particularly differentially expressed fragments,are useful in the study or treatment of conditions which are mediated byB cell activity, i.e., to diagnose, treat or prevent B cell-mediateddisorders. Thus, “B cell mediated disorders” or “disease states” caninclude conditions involving, for example, arthritis, diabetes, ormultiple sclerosis.

[0209] Methods of modulating B cell activity in cells or organisms areprovided. Some methods comprise administering to a cell ananti-differentially expressed antibody or other agent identified hereinor by the methods provided herein, that reduces or eliminates thebiological activity of the endogeneous differentially expressed protein.Alternatively, the methods comprise administering to a cell or organisma recombinant nucleic acid encoding a differentially expressed proteinor modulator including anti-sense nucleic acids. As will be appreciatedby those in the art, this can be accomplished in any number of ways. Insome preferred methods, the activity of differentially expressed isincreased by increasing the amount of differentially expressed in thecell, for example by overexpressing the endogeneous differentiallyexpressed or by administering a differentially expressed gene, usingknown gene therapy techniques, for example. In a preferred method, thegene therapy techniques include the incorporation of the exogenous geneusing enhanced homologous recombination (EHR), for example as describedin PCT/US93/03868, hereby incorporated by reference in its entirety.

[0210] In some methods, the invention provides methods for diagnosing anB cell activity related condition in an individual. The methods comprisemeasuring the activity of differentially expressed protein in a tissuefrom the individual or patient, which can include a measurement of theamount or specific activity of the protein. This activity is compared tothe activity of differentially expressed from either a unaffected secondindividual or from an unaffected tissue from the first individual. Whenthese activities are different, the first individual can be at risk foran B cell activity mediated disorder.

[0211] Furthermore, nucleotide sequences encoding a differentiallyexpressed protein can also be used to construct hybridization probes formapping the gene which encodes that differentially expressed protein andfor the genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein can be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

[0212] 13. Antibodies

[0213] In some methods, the differentially expressed proteins of thepresent invention can be used to generate polyclonal and monoclonalantibodies to differentially expressed proteins, which are useful asdescribed herein. A number of immunogens are used to produce antibodiesthat specifically bind differentially expressed polypeptides.Full-length differentially expressed polypeptides are suitableimmunogens. Typically, the immunogen of interest is a peptide of atleast about 3 amino acids, more typically the peptide is at least 5amino acids in length, preferably, the fragment is at least 10 aminoacids in length and more preferably the fragment is at least 15 aminoacids in length. The peptides can be coupled to a carrier protein (e.g.,as a fusion protein), or are recombinantly expressed in an immunizationvector. Antigenic determinants on peptides to which antibodies bind aretypically 3 to 10 amino acids in length. Naturally occurringpolypeptides are also used either in pure or impure form. Recombinantpolypeptides are expressed in eukaryotic or prokaryotic cells andpurified using standard techniques. The polypeptide, or a syntheticversion thereof, is then injected into an animal capable of producingantibodies. Either monoclonal or polyclonal antibodies can be generatedfor subsequent use in immunoassays to measure the presence and quantityof the polypeptide.

[0214] These antibodies find use in a number of applications. Forexample, the differentially expressed antibodies can be coupled tostandard affinity chromatography columns and used to purifydifferentially expressed proteins as further described below. Theantibodies can also be used as blocking polypeptides, as outlined above,since they will specifically bind to the differentially expressedprotein.

[0215] The anti-differentially expressed protein antibodies can comprisepolyclonal antibodies. Methods for producing polyclonal antibodies areknown to those of skill in the art. In brief, an immunogen, preferably apurified polypeptide, a polypeptide coupled to an appropriate carrier(e.g., GST and keyhole limpet hemocyanin), or a polypeptide incorporatedinto an immunization vector such as a recombinant vaccinia virus (see,U.S. Pat. No. 4,722,848) is mixed with an adjuvant and animals areimmunized with the mixture. The animal's immune response to theimmunogen preparation is monitored by taking test bleeds and determiningthe titer of reactivity to the polypeptide of interest. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to thepolypeptide is performed where desired. See, e.g., Coligan (1991)CURRENT PROTOCOLS IN IMMUNOLOGY Wiley/Greene, NY; and Harlow and Lane(1989) ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Press, NY.

[0216] Antibodies, including binding fragments and single chainrecombinant versions thereof, against predetermined fragments ofdifferentially expressed proteins are raised by immunizing animals,e.g., with conjugates of the fragments with carrier proteins asdescribed above.

[0217] The anti-differentially expressed protein antibodies can,alternatively, be monoclonal antibodies. The monoclonal antibodies areprepared from cells secreting the desired antibody. These antibodies arescreened for binding to normal or modified polypeptides, or screened foragonistic or antagonistic activity, e.g., activity mediated through thedifferentially expressed proteins. In some instances, it is desirable toprepare monoclonal antibodies from various mammalian hosts, such asmice, rodents, primates, and humans. Description of techniques forpreparing such monoclonal antibodies are found in, e.g., Stites et al.(eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lange MedicalPublications, Los Altos, Calif., and references cited therein; Harlowand Lane, Supra; Goding (1986) Monoclonal Antibodies: Principles andPractice (2d ed.) Academic Press, New York, N.Y.; and Kohler andMilstein (1975) Nature 256: 495-497.

[0218] The immunizing agent will typically include the differentiallyexpressed protein polypeptide or a fusion protein thereof. Generally,either peripheral blood lymphocytes (“PBLs”) are used if cells of humanorigin are desired, or spleen cells or lymph node cells are used ifnon-human mammalian sources are desired. The lymphocytes are then fusedwith an immortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

[0219] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

[0220] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst differentially expressed protein. Preferably, the bindingspecificity of monoclonal antibodies produced by the hybridoma cells isdetermined by immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem. 107:220 (1980).

[0221] After the desired hybridoma cells are identified, the clones canbe subdloned by limiting dilution procedures and grown by standardmethods (Goding, supra). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

[0222] The monoclonal antibodies secreted by the subclones can beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0223] Other suitable techniques involve selection of libraries ofrecombinant antibodies in phage or similar vectors. See, Huse et al.(1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341:544-546.

[0224] Also, recombinant immunoglobulins may be produced. See, U.S. Pat.No. 4,816,567 (Cabilly); and Queen et al. (1989) Proc. Nat'l Acad. Sci.USA 86: 10029-10033.

[0225] Briefly, nucleic acids encoding light and heavy chain variableregions, optionally linked to constant regions, are inserted intoexpression vectors. The light and heavy chains can be cloned in the sameor different expression vectors. The DNA segments encoding antibodychains are operably linked to control sequences in the expressionvector(s) that ensure the expression of antibody chains. Such controlsequences include a signal sequence, a promoter, an enhancer, and atranscription termination sequence. Expression vectors are typicallyreplicable in the host organisms either as episomes or as an integralpart of the host chromosome.

[0226]E. coli is one procaryotic host particularly for expressingantibodies of the present invention. Other microbial hosts suitable foruse include bacilli, such as Bacillus subtilus, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which typically contain expression control sequencescompatible with the host cell (e.g., an origin of replication) andregulatory sequences such as a lactose promoter system, a tryptophan(trp) promoter system, a beta-lactamase promoter system, or a promotersystem from phage lambda.

[0227] Other microbes, such as yeast, may also be used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired.

[0228] Mammalian tissue cell culture can also be used to express andproduce the antibodies of the present invention (See Winnacker, FromGenes to Clones (VCH Publishers, N.Y., 1987). Eukaryotic cells arepreferred, because a number of suitable host cell lines capable ofsecreting intact antibodies have been developed. Preferred suitable hostcells for expressing nucleic acids encoding the immunoglobulins of theinvention include: monkey kidney CV1 line transformed by SV40 (COS-7,ATCC CRL 1651); human embryonic kidney line (293) (Graham et al., 1977,J. Gen. Virol. 36:59); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin, 1980, Proc.Natl. Acad. Sci. U.S.A. 77:4216); mouse sertoli cells (TM4, Mather,1980, Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70);african green monkey kidney cells (VERO-76, ATCC CRL 1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); and, TRI cells (Mather etal., 1982, Annals N.Y. Acad. Sci. 383:44-46); baculovirus cells.

[0229] The vectors containing the polynucleotide sequences of interest(e.g., the heavy and light chain encoding sequences and expressioncontrol sequences) can be transferred into the host cell. Calciumchloride transfection is commonly utilized for prokaryotic cells,whereas calcium phosphate treatment or electroporation can be used forother cellular hosts. (See generally Sambrook et al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989)(incorporated by reference in its entirety for all purposes). When heavyand light chains are cloned on separate expression vectors, the vectorsare co-transfected to obtain expression and assembly of intactimmunoglobulins. After introduction of recombinant DNA, cell linesexpressing immunoglobulin products are cell selected. Cell lines capableof stable expression are preferred (i.e., undiminished levels ofexpression after fifty passages of the cell line).

[0230] Once expressed, the whole antibodies, their dimers, individuallight and heavy chains, or other immunoglobulin forms of the presentinvention can be purified according to standard procedures of the art,including ammonium sulfate precipitation, affinity columns, columnchromatography, gel electrophoresis and the like (See generally Scopes,Protein Purification (Springer-Verlag, N.Y., 1982). Substantially pureimmunoglobulins of at least about 90 to 95% homogeneity are preferred,and 98 to 99% or more homogeneity most preferred.

[0231] Frequently, the polypeptides and antibodies will be labeled byjoining, either covalently or non-covalently, a substance which providesfor a detectable signal. A wide variety of labels and conjugationtechniques are known and are reported extensively in both the scientificand patent literature. Thus, an antibody used for detecting an analytecan be directly labeled with a detectable moiety, or may be indirectlylabeled by, for example, binding to the antibody a secondary antibodythat is, itself directly or indirectly labeled.

[0232] The antibodies of this invention are also used for affinitychromatography in isolating differentially expressed proteins. Columnsare prepared, e.g., with the antibodies linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysateis passed through the column, washed, and treated with increasingconcentrations of a mild denaturant, whereby purified differentiallyexpressed polypeptides are released.

[0233] A further approach for isolating DNA sequences which encode ahuman monoclonal antibody or a binding fragment thereof is by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al, Science 246:1275-1281 (1989) and then cloningand amplifying the sequences which encode the antibody (or bindingfragment) of the desired specificity. Such B cells can be obtained froma human immunized with the desired antigen, fragments, longerpolypeptides containing the antigen or fragments or anti-idiotypicantibodies. Optionally, such B cells are obtained from an individual whohas not been exposed to the antigen. B cell can also be obtained fromtransgenic non-human animals expressing human immunoglobulin sequences.The transgenic non-human animals can be immunized with an antigen orcollection of antigens. The animals can also be unimmunized. B cell mRNAsequences encoding human antibodies are used to generate cDNA usingreverse transcriptase. The V region encoding segments of the cDNAsequences are then cloned into a DNA vector that directs expression ofthe antibody V regions. Typically the V region sequences arespecifically amplified by PCR prior to cloning. Also typically, the Vregion sequences are cloned into a site within the DNA vector that isconstructed so that the V region is expressed as a fusion protein.Examples of such fusion proteins include ml 3 coliphage gene 3 and gene8 fusion proteins. The collection of cloned V region sequences is thenused to generate an expression library of antibody V regions. Togenerate an expression library, the DNA vector comprising the cloned Vregion sequences is used to transform eukaryotic or prokaryotic hostcells. In addition to V regions, the vector can optionally encode all orpart of a viral genome, and can comprise viral packaging sequences. Insome cases the vector does not comprise an entire virus genome, and thevector is then used together with a helper virus or helper virus DNAsequences. The expressed antibody V regions are found in, or on thesurface of, transformed cells or virus particles from the transformedcells. This expression library, comprising the cells or virus particles,is then used to identify V region sequences that encode antibodies, orantibody fragments reactive with predetermined antigens. To identifythese V region sequences, the expression library is screened or selectedfor reactivity of the expressed V regions with the predeterminedantigens. The cells or virus particles comprising the cloned V regionsequences, and having the expressed V regions, are screened or selectedby a method that identifies or enriches for cells or virus particlesthat have V regions reactive (e.g., binding association or catalyticactivity) with a predetermined antigen. For example, radioactive orfluorescent labeled antigen that then binds to expressed V regions canbe detected and used to identify or sort cells or virus particles.Antigen bound to a solid matrix or bead can also be used to select cellsor virus particles having reactive V regions on the surface. The Vregion sequences thus identified from the expression library can then beused to direct expression, in a transformed host cell, of an antibody orfragment thereof, having reactivity with the predetermined antigen.

[0234] The protocol described by Huse is rendered more efficient incombination with phage-display technology. See, e.g., Dower et al., WO91/17271 and McCafferty et al., WO 92/01047, U.S. Pat. Nos. 5,871,907,5,858,657, 5,837,242, 5,733,743 and 5,565,332 (each of which isincorporated by reference in its entirety for all purposes). In thesemethods, libraries of phage are produced in which members (displaypackages) display different antibodies on their outer surfaces.Antibodies are usually displayed as Fv or Fab fragments. Phagedisplaying antibodies with a desired specificity can be selected byaffinity enrichment to the antigen or fragment thereof. Phage displaycombined with immunized transgenic non-human animals expressing humanimmunoglobulin genes can be used to obtain antigen specific antibodieseven when the immune response to the antigen is weak.

[0235] In a variation of the phage-display method, human antibodieshaving the binding specificity of a selected murine antibody can beproduced. See, for example, Winter, WO 92/20791. In this method, eitherthe heavy or light chain variable region of the selected murine antibodyis used as a starting material. If, for example, a light chain variableregion is selected as the starting material, a phage library isconstructed in which members display the same light chain variableregion (i.e., the murine starting material) and a different heavy chainvariable region. The heavy chain variable regions are obtained from alibrary of rearranged human heavy chain variable regions. A phageshowing strong specific binding (e.g., at least 10⁸ and preferably atleast 10⁹ M⁻¹) can then be selected. The human heavy chain variableregion from this phage then serves as a starting material forconstructing a further phage library. In this library, each phagedisplays the same heavy chain variable region (i.e., the regionidentified from the first display library) and a different light chainvariable region. The light chain variable regions are obtained from alibrary of rearranged human variable light chain regions. Again, phageshowing strong specific binding for the selected are selected.Artificial antibodies that are similar to human antibodies can beobtained from phage display libraries that incorporate random orsynthetic sequences, for example, in CDR regions.

[0236] In another embodiment of the invention, fragments of antibodiesagainst differentially expressed protein or protein analogs areprovided. Typically, these fragments exhibit specific binding to thedifferentially expressed protein receptor similar to that of a completeimmunoglobulin. Antibody fragments include separate heavy chains, lightchains F_(ab), F_(ab′) F_((ab′)2) and F_(v). Fragments are produced byrecombinant DNA techniques, or by enzymic or chemical separation ofintact immunoglobulins.

[0237] The antibodies can be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0238] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art.

[0239] An alternative approach is the generation of humanizedimmunoglobulins by linking the CDR regions of non-human antibodies tohuman constant regions by recombinant DNA techniques. See U.S. Pat. No.5,585,089 (Queen et al.). Humanized forms of non-human (e.g., murine)antibodies are immunoglobulins, immunoglobulin chains or fragmentsthereof (such as F_(v), F_(ab), F_(ab′), F_(ab2) or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies can also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of anFc region, typically that of a human immunoglobulin (Jones et al.,Nature (1986) 321:522-525; Riechmann et al, Nature (1988) 332:323-329;and Presta, Curr. Op. Struct. Biol. (1992) 2: 593-596).

[0240] Chimeric and humanized antibodies have the same or similarbinding specificity and affinity as a mouse or other nonhuman antibodythat provides the starting material for construction of a chimeric orhumanized antibody. Chimeric antibodies are antibodies whose light andheavy chain genes have been constructed, typically by geneticengineering, from immunoglobulin gene segments belonging to differentspecies. For example, the variable (V) segments of the genes from amouse monoclonal antibody may be joined to human constant (C) segments,such as IgG, and IgG₄. Human isotype IgG, is preferred. A typicalchimeric antibody is thus a hybrid protein consisting of the V orantigen-binding domain from a mouse antibody and the C or effectordomain from a human antibody.

[0241] Humanized antibodies have variable region framework residuessubstantially from a human antibody (termed an acceptor antibody) andcomplementarity determining regions substantially from a mouse-antibody(referred to as the donor iimmunoglobulin). See, Queen et al., 1989,Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033 and WO 90/07861, U.S. Pat.No. 5,693,762, U.S. 5,693,761, U.S. 5,585,089, U.S. 5,530,101 andWinter, U.S. Pat. No. 5,225,539 (incorporated by reference in theirentirety for all purposes). The constant region(s), if present, are alsosubstantially or entirely from a human immunoglobulin. The humanvariable domains are usually chosen from human antibodies whoseframework sequences exhibit a high degree of sequence identity with themurine variable region domains from which the CDRs were derived. Theheavy and light chain variable region framework residues can be derivedfrom the same or different human antibody sequences. The human antibodysequences can be the sequences of naturally occurring human antibodiesor can be consensus sequences of several human antibodies. See Carter etal., WO 92/22653. Certain amino acids from the human variable regionframework residues are selected for substitution based on their possibleinfluence on CDR conformation and/or binding to antigen. Investigationof such possible influences is by modeling, examination of thecharacteristics of the amino acids at particular locations, or empiricalobservation of the effects of substitution or mutagenesis of particularamino acids.

[0242] For example, when an amino acid differs between a murine variableregion framework residue and a selected human variable region frameworkresidue, the human framework amino acid should usually be substituted bythe equivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid: (1) noncovalently binds antigendirectly, (2) is adjacent to a CDR region, (3) otherwise interacts witha CDR region (e.g. is within about 6 A of a CDR region), or (4)participates in the VL-VH interface.

[0243] Other candidates for substitution are acceptor human frameworkamino acids that are unusual for a human immunoglobulin at thatposition. These amino acids can be substituted with amino acids from theequivalent position of the mouse donor antibody or from the equivalentpositions of more typical human immunoglobulins. Other candidates forsubstitution are acceptor human framework amino acids that are unusualfor a human immunoglobulin at that position. The variable regionframeworks of humanized immunoglobulins usually show at least 85%sequence identity to a human variable region framework sequence orconsensus of such sequences.

[0244] Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries discussed above(Hoogenboom and Winter, J. Mol. Biol. (1991) 227: 381; Marks et al., J.Mol. Biol. (1991) 222: 581). The techniques of Cole et al. and Boerneret al. are also available for the preparation of human monoclonalantibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, p. 77 (1985) and Boerner et al., J. Immunol. (1991) 147(1):86-95). Similarly, human antibodies can be made by introducing of humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016; see also Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature (1994) 368:856-859; Morrison, Nature (1994) 368: 812-13; Fishwild et al., NatureBiotechnology (1996) 14: 845-51; Neuberger, Nature Biotechnology (1996)14: 826; Lonberg and Huszar, Intern. Rev. Immunol. (1995) 13: 65-93.

[0245] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for the differentially expressed protein, the other oneis for any other antigen, and preferably for a cell-surface protein orreceptor or receptor subunit.

[0246] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities Milsteinand Cuello, Nature (1983) 305: 537-539). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J. (1991)10:3655-3659.

[0247] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology (1986) 121:210.

[0248] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

[0249] The anti-differentially expressed protein antibodies of theinvention have various utilities. For example, anti-differentiallyexpressed protein antibodies can be used in diagnostic assays for adifferentially expressed protein, e.g., detecting its expression inspecific cells, tissues, or serum. Various diagnostic assay techniquescan be used, such as competitive binding assays, direct or indirectsandwich assays and immunoprecipitation assays conducted in eitherheterogeneous or homogeneous phases (Zola, Monoclonal Antibodies: AManual of Techniques, CRC Press, Inc. (1987) pp. 147-158). Theantibodies used in the diagnostic assays can be labeled with adetectable moiety. The detectable moiety should be capable of producing,either directly or indirectly, a detectable signal. For example, thedetectable moiety can be a radioisotope, such as 3H, ¹⁴c, ³²P, ³⁵S, or¹²⁵I, a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkalinephosphatase, betagalactosidase or horseradish peroxidase. Any methodknown in the art for conjugating the antibody to the detectable moietycan be employed, including those methods described by Hunter et al.,Nature (1962) 144: 945; David et al., Biochemistry (1974) 13:1014; Painet al., J. Immunol. Meth. (1981) 40:219; and Nygren, J. Histochem. andCytochem. (1982) 30:407.

[0250] Anti-differentially expressed protein antibodies also are usefulfor the affinity purification of differentially expressed protein fromrecombinant cell culture or natural sources. In this process, theantibodies against differentially expressed protein are immobilized on asuitable support, such a Sephadex resin or filter paper, using methodswell known in the art. The immobilized antibody then is contacted with asample containing the differentially expressed protein to be purified,and thereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except thedifferentially expressed protein, which is bound to the immobilizedantibody. Finally, the support is washed with another suitable solventthat will release the differentially expressed protein from theantibody.

[0251] 14. Pharmaceutical Compositions and Methods of Administration

[0252] The anti-differentially expressed protein antibodies can also beused in treatment. In some methods, the genes encoding the antibodiesare provided, such that the antibodies bind to and modulate thedifferentially expressed protein within the cell. In other methods, atherapeutically effective amount of a differentially expressed protein,agonist or antagonist is administered to a patient. A “therapeuticallyeffective amount”, “pharmacologically acceptable dose”,“pharmacologically acceptable amount” means that a sufficient amount ofan immunosuppressive agent or combination of agents is present toachieve a desired result, e.g., preventing, delaying, inhibiting orreversing a symptom of a disease or disorder or the progression ofdisease or disorder when administered in an appropriate regime.

[0253] Pharmaceutically acceptable carriers are determined in part bythe particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention (see, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed., 1989). The pharmaceuticalcompositions of the present invention generally comprise adifferentially expressed protein, agonist or antagonist in a formsuitable for administration to a patient. The pharmaceuticalcompositions are generally formulated as sterile, substantially isotonicand in full compliance with all Good Manufacturing Practice (GMP)regulations of the U.S. Food and Drug Administration.

[0254] Formulations suitable for oral administration can consist of (a)liquid solutions, such as an effective amount of the packaged nucleicacid suspended in diluents, such as water, saline or PEG 400; (b)capsules, sachets or tablets, each containing a predetermined amount ofthe active ingredient, as liquids, solids, granules or gelatin; (c)suspensions in an appropriate liquid; and (d) suitable emulsions. Tabletforms can include one or more of lactose, sucrose, mannitol, sorbitol,calcium phosphates, corn starch, potato starch, microcrystallinecellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate,stearic acid, and other excipients, colorants, fillers, binders,diluents, buffering agents, moistening agents, preservatives, flavoringagents, dyes, disintegrating agents, and pharmaceutically compatiblecarriers. Lozenge forms can comprise the active ingredient in a flavor,usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin or sucrose and acacia emulsions, gels, and the like containing,in addition to the active ingredient, carriers known in the art.

[0255] In some preferred methods, the pharmaceutical compositions are ina water soluble form, such as being present as pharmaceuticallyacceptable salts, which is meant to include both acid and base additionsalts. “Pharmaceutically acceptable acid addition salt” refers to thosesalts that retain the biological effectiveness of the free bases andthat are not biologically or otherwise undesirable, formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, maleic acid, malonic acid, succinic acid, flmaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Particularly preferred are theammonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

[0256] The nucleic acids, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

[0257] Suitable formulations for rectal administration include, forexample, suppositories, which consist of the packaged nucleic acid witha suppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons. In addition, it isalso possible to use gelatin rectal capsules which consist of acombination of the packaged nucleic acid with a base, including, forexample, liquid triglycerides, polyethylene glycols, and paraffinhydrocarbons.

[0258] Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and nonaqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.Formulations for injection can be presented in unit dosage form, e.g.,in ampules or in multidose containers, with an added preservative. Thecompositions are formulated as sterile, substantially isotonic and infall compliance with all Good Manufacturing Practice (GMP) regulationsof the U.S. Food and Drug Administration.

[0259] Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by the packaged nucleic acid as described above in thecontext of ex vivo therapy can also be administered intravenously orparenterally as described above.

[0260] The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

[0261] In determining the effective amount of the vector to beadministered in the treatment or prophylaxis of conditions resultingfrom expression of the differentially expressed proteins of theinvention, the physician evaluates circulating plasma levels of thevector, vector toxicities, progression of the disease, and theproduction of anti-vector antibodies. In general, the dose equivalent ofa naked nucleic acid from a vector is from about 1 μg to 100 μg for atypical 70 kilogram patient, and doses of vectors which include aretroviral particle are calculated to yield an equivalent amount oftherapeutic nucleic acid.

[0262] For administration, inhibitors and transduced cells of thepresent invention can be administered at a rate determined by the LD₅₀of the inhibitor, vector, or transduced cell type, and the side-effectsof the inhibitor, vector or cell type at various concentrations, asapplied to the mass and overall health of the patient. Administrationcan be accomplished via single or divided doses.

[0263] Transduced cells are prepared for reinfusion according toestablished methods (see Abrahamsen et al., J. Clin. Apheresis 6:48-53(1991); Carter et al., J. Clin. Arpheresis 4:113-117 (1998); Aebersoldetal., J. Immunol. Meth. 112:1-7 (1998); Muul et al., J. Immunol.Methods 101:171-181 (1987); and Carter et al., Transfusion 27:362-365(1987)). After a period of about 2-4 weeks in culture, the cells shouldnumber between 1×10⁸ and 1×10¹². In this regard, the growthcharacteristics of cells vary from patient to patient and from cell typeto cell type. About 72 hours prior to reinfusion of the transducedcells, an aliquot is taken for analysis of phenotype, and percentage ofcells expressing the therapeutic agent.

[0264] 15. Kits

[0265] The differentially expressed protein, agonist or antagonist ofthe present invention or their homologs are useful tools for examiningexpression and regulation of Sbo family potassium channels. Reagentsthat specifically hybridize to nucleic acids encoding differentiallyexpressed proteins of the invention (including probes and primers of thedifferentially expressed proteins), and reagents that specifically bindto the differentially expressed proteins, e.g., antibodies, are used toexamine expression and regulation.

[0266] Nucleic acid assays for the presence of differentially expressedproteins in a sample include numerous techniques are known to thoseskilled in the art, such as Southern analysis, northern analysis, dotblots, RNase protection, SI analysis, amplification techniques such asPCR and LCR, high density oligonucleotide array analysis, and in situhybridization. In in situ hybridization, for example, the target nucleicacid is liberated from its cellular surroundings in such as to beavailable for hybridization within the cell while preserving thecellular morphology for subsequent interpretation and analysis. Thefollowing articles provide an overview of the art of in situhybridization: Singer et al., Biotechniques 4:230-250 (1986); Haase etal., Methods in Virology, vol. VII, pp. 189-226 (1984); and Nucleic AcidHybridization: A Practical Approach (Hamnes et al., eds. 1987). Inaddition, a differentially expressed protein can be detected with thevarious immunoassay techniques described above. The test sample istypically compared to both a positive control (e.g., a sample expressingrecombinant differentially expressed protein) and a negative control.

[0267] The present invention also provides for kits for screening B cellactivity modulators. Such kits can be prepared from readily availablematerials and reagents. For example, such kits can comprise any one ormore of the following materials: the differentially expressed proteins,agonists, or antagonists of the present invention, reaction tubes, andinstructions for testing the activities of differentially expressedgenes.

[0268] A wide variety of kits and components can be prepared accordingto the present invention, depending upon the intended user of the kitand the particular needs of the user. For example, the kit can betailored for in vitro or in vivo assays for measuring the activity of adifferentially expressed proteins or B cell activity modulators of thepresent invention.

[0269] The invention further provides kits comprising probe arrays asdescribed above. Optional additional components of the kit include, forexample, other restriction enzymes, reverse-transcriptase or polymerase,the substrate nucleoside triphosphates, means used to label (forexample, an avidin-enzyme conjugate and enzyme substrate and chromogenif the label is biotin), and the appropriate buffers for reversetranscription, PCR, or hybridization reactions.

[0270] Usually, the kits of the present invention also containinstructions for carrying out the methods.

EXAMPLES

[0271] Methods

[0272] B Cell Purification and Stimulation

[0273] Splenic B cells from non-transgenic, Ig^(HEL) or sHEL/Ig^(HEL)transgenic mice were purified at room temperature in 1% bovine calfserum in RPMI. The spleen cells were stained with CD4, CD8 and Mac-1FITC conjugated antibodies (Caltag) and depleted of T cells andmacrophages with sheep anti-FITC magnetic beads (Perseptive Biosystems).The remaining cells were 85-95% B220 positive and were either lysedimmediately (naive and tolerant cell preps) or stimulated in RPMI with1% serum at 37° C. at 2-3×10⁶ cells/ml. For stimulation experiments, HEL(Sigma) was used at 500 ng/ml, goat anti-mu (Jackson Labs) at 10 μg/ml,FK506 at 10 ng/ml, PD98059 (NEB) at 20 μM unless stated otherwise,ionomycin at 1 μM and EGTA at 3 mM. Cells were preincubated for 45minutes with PD98059, 15 minutes with FK506 and 2 minutes with EGTAbefore addition of HEL or anti-mu. Mock stimulations were performed byaddition of carrier alone for stimuli or inhibitors. At the end of theincubation, the cells were pelleted by centrifugation, resuspended in aminimal volume of medium (about 50 μl) by pipetting and lysed in 0.5-1ml Trizol (Gibco BRL).

[0274] Naïve and tolerant B cells were also purified by FACS. Spleencells from Ig^(HEL) and sHEL/Ig^(HEL) mice were stained for B220 andCD21 and sorted for B220 positive, CD21^(medium) cells. Marginal zonecells (CD21 high) were excluded from the gate. FACS allowed us tocontrol for the fact that B cells in sHEL/Ig^(HEL) mice are generallypresent at lower numbers than Ig^(HEL) mice and the marginal zone B cellsubset is absent. Thus, expression changes between anergic and naivecells determined from samples purified by negative depletion that arealso seen in cells purified by FACS are unlikely to be due to systematicdifferences in the amount of marginal zone B cells or non-B cells in thetwo samples.

[0275] RNA Purification, cDNA Synthesis, In Vitro Transcription (IVT)and Array Hybridization

[0276] Trizol lysates were phenol-extracted and precipitated withisopropanol. Poly A+ RNA was purified with Oligotex (Qiagen). cDNA wassynthesised with a Superscriptll cDNA synthesis kit (Gibco BRL) using aT7:(dT)24 oligo to prime the first strand and purified by phenolextraction and ethanol precipitation. The cDNA was used as a templatefor in vitro transcription (IVT) using the Megascript system (Ambion)with the inclusion of biotinylated CTP and UTP. The IVT product wasseparated from unincorporated nucleotides using a RNeasy column (Qiagen)and was fragmented with 150 mM magnesium chloride at 95° C. FragmentedcRNA was hybridised in a volume of 200 pl 1 M sodium chloride, 10 mMTris pH 7.4, 0.005% Triton X100 including 1 mg/ml BSA, 0.1 mg/ml herringsperm DNA and bacterial transcripts spiked at known concentrations.Hybridization was for 12-16 hours at 40° C. with rotation. The arrayswere incubated at 50° C. for 1 hr then washed to 0.5X SSPE, roomtemperature. Biotinylated hybridised cRNA was developed by staining withstrepavidin-PE and the chips were scanned with a Molecular Dynamicsscanner (see also Lipshutz, R. J. et al., Nat. Genet (1999) 21: 20-4;this reference and references cited therein are herein incorporated byreference).

[0277] Statistical Analysis and Querying

[0278] Each gene on the arrays was tiled as a collection ofapproximately 20 probe pairs. Each probe pair contains a 25mer oligothat is exactly complementary to the transcript (the perfect matcholigo) and a second oligo that contains a single base mismatch at themiddle base. The perfect match—mismatch intensities (PM-MM) werecalculated for each probe pair for each gene in each experiment. Foreach probe pair, the PM-MM values were compared by t test between thetwo conditions (e.g. resting vs stimulated, matched t test; naive vsanergic, unmatched t test). Positive t values associated with aprobability of less than 0.1 were scored as increased, negative t valueswith a probability of less than 0.1 were scored as decreased. The numberof increased and decreased probe pairs associated with each gene wasdetermined and called npos and nneg respectively (of the approximately20 probe pairs tiled for each gene). The distribution of npos and nnegis binomial where p=0.1 and n=number of probe pairs for that gene. Theprobability of scoring npos or more of the total number of probe pairswas determined. The same analysis was done for decreased genes usingnneg. A probability value was chosen so that the probability of one ormore false positive in any query was less than 5% after correcting forthe number of genes queried. For example, 280 genes had a median foldchange after 1 hour activation of 1.75 or greater. The adjusted 5%probability for 280 trials is 0.00018, and 59 of the 280 changes weresignificant at this level. We also analysed our data using an analysisof variance approach where the variation in PM-MM values for each genewas partitioned into that due to error (within group), probe pair, orthe experimental factor (e.g. resting vs stimulated). The results werebroadly the same as for the t test strategy described above. However,the t test strategy is more robust to rare probes that had very high andanomalous signals.

[0279] To analyse the 6 hr timepoint (2 experiments) and the sortednaive and tolerant B cells we used the following query. For a giventranscript, each probe pair was scored as increased in sample A relativeto sample B if (PM-MM)_(A)-(PM-MM)_(B)>30 and(PM-MM)_(A)>1.3×(PM-MM)_(B) and decreased by the reverse. A transcriptwas scored as increased in each pairwise comparison if the number ofincreased probe pairs was 3 or greater, the ratio of increased todecreased probe pairs was greater than 3 and the ratio of averagedifference intensities was greater than 1.8. Decreased genes weredetermined by the reverse of this algorithm. Based on comparisons of allgenes between closely matched samples the false positive rate of thisquery was empirically determined to be approximately 1 in 18 transcriptsin any pairwise comparison. Consistent changes across the 2 experimentshave a false positive rate of approximately 1 in 300.

[0280] Measurement of Gene Expression

[0281] An intensity for each gene was calculated based on a trimmed meanof the PM-MM values. Values less than 5 were consideredindistinguishable and were set to 5. The resulting average differenceintensities were used to represent expression levels in the figures andto calculate fold changes.

[0282] Results

[0283] To identify molecular events distinguishing activation andtolerance in peripheral lymphocytes, gene expression profiles wereanalyzed in lymphocytes undergoing these opposing processes that were inall other respects as closely matched as possible. Homogeneouspopulations of B lymphocytes specific for a well defined antigen, henegg lysozyme (HEL), were obtained from the spleen of mice transgenic fora B cell antigen receptor against HEL (Ig^(HEL) mice). Resting B cellsfrom Ig^(HEL) mice are antigenically naive and in G0 of cell cycle.Acute stimulation with foreign HEL triggers their activation and entryinto G1, promoting clonal proliferation and antibody secretion providedthat T cells or bacterial lipopolysaccharides are present as costimuli.In parallel, homogeneous populations of self-tolerant (anergic) B cellswere obtained from the spleen of double transgenic mice which carry thesame Ig^(HEL) receptor transgene but also express HEL as a self antigen(sHEL:Ig^(HEL) mice). Despite expressing the same HEL-specific receptorsand being matched for stage of development, the tolerant cells areunable to make a proliferative or antibody response to HEL. Instead,repeated stimulation of their receptors by self HEL causes them to makeresponses that actively reinforce tolerance, such as altered migration,Fas-dependent and Fas-independent apoptosis, and inhibition of plasmacell differentiation. Peripheral tolerant B cells in the sHEL:Ig^(HEL)mice first encountered antigen during development in the bone marrow andhave a life span of about 2 weeks. Despite the fact that these cellshave been exposed to self-antigen for differing lengths of time, theyappear homogenous with respect to the aspects of tolerance listed above,and as measured by continuous calcium oscillations and uniformly lowexpression of the activation markers B7-2 and CD69.

[0284] 6,500 genes for changes in expression during activation ortolerance using a set of Affymetrix GeneChip™ expression arrays(reviewed in Lipshutz et al.). Hybridization intensity levels correspondonly approximately to absolute expression levels as the protocol relieson an amplification scheme that can not be strictly quantitative for alltranscripts. However, relative expression levels of the same transcriptsacross different samples are conserved and absolute levels are accuratewithin +/−2 fold.

[0285] A relatively small set of response genes was associated with theinitial phase of B lymphocyte activation, one hour after foreign antigenstimulation ex vivo (FIG. 1). Of the 6,500 genes screened in sevenindependent replicate experiments, mRNAs for only thirty seven weresignificantly increased and twenty two were decreased (FIG. 1,p<0.00018). A large fraction of the increased and decreased transcriptsencode transcriptional regulators. While a small number of these 59transcripts have been previously identified as early response genes in Bcells, validating the data obtained here, most were not previously knownto participate in B cell responses. Many of these genes encode proteinswith established roles in mitotic and anti-apoptotic responses bylymphocytes or other cell types. For example, LSIRF is necessary formitogenesis as B cells from mice deficient in this gene are unable toproliferate in response to anti-IgM. Furthermore, expression of A1, abcl-2 homologue, can be sufficient to prevent apoptosis after antigenreceptor engagement on B cells. Conversely, down-regulation of LKLF canbe obligatory for B cell activation as T cells deficient in LKLF have aspontaneously activated cell surface phenotype. Finally, c-myc, c-fos,and FosB are associated with mitogenesis through their status asoncogenes and Egr-1 and Egr-2 have been specifically implicated in Bcell mitogenesis.

[0286] The pattern of gene expression was much more extensively alteredafter six hours of antigen stimulation. While mRNAs for many of the 1 hrinduced genes had decreased by this time (e.g., Egr-1, PAC-1, c-fos,FosB in FIG. 1C), others in the early response set showed sustained orexaggerated responses at 6 hours (e.g., A1 and MIP-1a/b in FIG. 1C, LKLFand GILZ in FIG. 1D). Many of the additional gene expression changes areconsistent with movement to the G1 phase of the cell cycle, includingupregulation of CDK4 and cyclin D2.

[0287] The same set of 6,500 genes was screened for expression changesin anergic B cells undergoing peripheral tolerance responses to HELantigen in vivo. Expression was compared between five tolerant B cellpreparations from sHEL:Ig^(HEL) mice and four naive B cell preparationsfrom Ig^(HEL) mice, where paired samples were purified by negativeselection with magnetic beads. A further two preparations each oftolerant and naive B cells were purified by positive selection on afluoresence activated cell sorter (FACS). Using an algorithm thatrequires consistency between both purification methods, expression ofonly twenty genes was significantly increased and eight genes decreasedin tolerant cells (FIG. 2, p<0.00034). One of these changes can be dueto contaminating erythroid cells (carbonic anhydrase II) because thismRNA species was much less abundant in FACS-sorted B cells.

[0288] To determine the extent of overlap between the responses to thesame antigen when presented as self or foreign the data was comparedusing both stringent (p value corrected for number of trials) andnon-stringent (uncorrected) queries. Of the 19 genes upregulated byself-antigen (excluding carbonic anhydrase II), 7 were also upregulatedby foreign antigen after 1 hour (p<0.00018) or 6 hours (2 of 2experiments): NAB2 and neurogranin were comparably upregulated by bothforms of antigen; Egr-1, Egr-2, Gfi-1, cyclin D2 and Cctq wereupregulated to a greater extent by foreign antigen than by self-antigen.Of the remaining 12 transcripts upregulated in tolerant cells, there wasweaker evidence for upregulation of 4 genes after 6 hours exposure toforeign antigen (SATB1, CD83, TGIF and CD72, 1 of 2 experiments). For 8of the 19 transcripts upregulated by self-antigen, there was no evidencefor upregulation by foreign antigen. Seven of 8 transcriptsdownregulated by self-antigen were downregulated by 6 hours exposure toforeign antigen in 2 of 2 experiments (4 transcripts) or 1 of 2experiments (3 transcripts). In summary, most, but not all, of thetranscript changes induced by selfantigen were also regulated by foreignantigen, though to differing degrees.

[0289] Only 16 of more than 500 transcript changes caused by foreignantigen were also regulated by self-antigen (p<0.05, fold change>1.8, atleast 1 of 2 experiments with sorted cells): nearly all of the responseto foreign antigen is blocked in tolerant cells. The response to foreignantigen is measured after in vitro stimulation whereas the response toself-antigen occurs in vivo. This was necessary because of technicallimitations on the length of time required to isolate and purifyactivated cells after stimulation in vivo relative to the time ofactivation. However, an analysis of transcript changes caused by invitro incubation in the absence of antigen is not consistent with thiscausing a partial activation response: in fact, some of the genes thatwere upregulated by antigen are downregulated by in vitro incubation andvice vers. Therefore, the differences that described between exposure toself and foreign antigen can reflect biological differences betweentolerance and immunity rather than an “adjuvant” effect of in vitroincubation.

[0290] FK506, a commonly used immunosuppressant drug, can block B cellactivation and can be a phenocopy of tolerance. B cells were stimulatedas for FIG. 1 but in the presence of 10 ng/ml FK506. This concentrationwas chosen as it is within the range maintained in the blood of kidneyand liver transplant patients receiving FK506 (also called Tacrolimusand Prograf, information on dosing fromhttp://www.fujisawa.com/info/medinfo/mnpginst.htm). Of the 59 genesdefined previously as increasing or decreasing 1 hr after B lymphocyteactivation, only one third of these were efficiently suppressed by thisdose of FK506 (FIG. 3). Some early response genes (for example, gadd153)were superinduced in the presence of drug. By this analysis, thesuppressive effects of FK506 on lymphocyte activation are much morelimited than the suppression achieved by peripheral tolerance. Theresponse genes blocked by the drug include genes that are triggered byself-antigen, such as Egr-2 and CD72, which can contribute to the activemaintenance of tolerance. Cells stimulated in the presence of FK506 donot activate NFAT, NFkB nor JNK though signaling through Erk is intact.Self-antigen causes apparent activation of more signaling pathways, assignaling through both Erk and NFAT is intact, but the response toantigen measured by transcript profiling is much more repressed than isachieved by FK506.

[0291] Naïve cells were stimulated in the presence of EGTA. This reagenthad essentially the same effect on the transcript profile as FK506 (FIG.3C), confirming that FK506 affected transcript levels through acalcium/calcineurin-dependent pathway. A notable exception to this wasMyD 116. Antigen induced upregulation of this transcript was repressedby FK506 (n=5) but not by EGTA (n=2), which can be indicative ofsecondary effects of the drug other than calcineurin inhibition.However, there were no FK506-induced changes in transcripts other thanthose altered in the antigen activation response.

[0292] The effects of tolerance and pharmacological reagents on B cellactivation can be used to assign transcriptional events downstream ofparticular signaling pathways (FIG. 4A). Transcriptional events that aresuppressed by FK506 and EGTA after antigen stimulation of naive cellscan be firmly assigned to be downsteam of the calcium/calcineurinpathway. These can be further subdivided on the basis of theirexpression levels in tolerant cells. In these cells, self-antigen evokeschronic low calcium oscillations that are sufficient to induce nucleartranslocation of NFAT but not to activate NFKB nor JNK, though all threepathways are dependent on calcium/calcineurin. Thus, FK506-sensitiveupregulation of genes not altered in tolerant cells is suggestive ofsignaling through NFKB or JNK, whereas FK506-sensitive genes that arealso upregulated in tolerance would be expected to be downstream of NFAT(for example, Egr-2 and CD72). Upregulation of Al is FK506 sensitive butblocked in tolerance.

[0293] In addition to NFAT, the ERK pathway is also activated in foreignor self antigen-stimulated cells. To determine downstreamtranscriptional effects of ERK, the effect of MEK, an ERK kinase, on Bcell activation was determined. The MEK inhibitor PD98059 was titratedin B cell activation experiments and gene expression was monitored usingone of the four arrays in the set (approximately 1600 genes).Upregulation of Egr-1 was totally inhibited by 20 μM PD98059 and was 50%inhibited at 5-10 μM, consistent with the potency of PD98059 againstrecombinant MEK (FIG. 3D). Regulation of other early response genes wasless sensitive than Egr-1. Induction of three transcripts (Egr-1, NAB2and Gfi-1) that are upregulated by both self and foreign antigen wassensitive to PD98059 (FIG. 3D) but insensitive to FK506 (FIG. 3A).Continuous activation of the ERK pathway by self-antigen can havetranscriptional consequences which are distinct from those downstream ofNFAT (FIG. 4A).

[0294] The strategy followed here, statistically comparing theexpression of large numbers of genes in replicate cell samples that wereclosely matched to eliminate secondary effects, provides the firstmolecular picture of how self-tolerance prevents lymphocyte mitogenesis(FIG. 4B). Given the continuous signaling through the ERK and NFATpathways in response to self antigen, it is remarkable how few of themitogenic response genes are triggered. The loss of a mitotic responseto antigen in tolerant cells is explained by the failure to upregulateLSIRF, a B cell myeloma protooncogene that is an essential transcriptionfactor for B cell mitogenic responses, and failure to upregulate A1, ananti-apoptotic protein that is sufficient and apparently necessary toblock apoptotic responses to antigen in B cells. The block to inductionof the B cell lymphoma protooncogene, c-myc, is also likely tocontribute since increased expression of c-myc is sufficient to promoteB cell blastogenesis in transgenic mice. By comparison, it is surprisinghow little of the early mitogenic response is suppressed by FK506 givenits ability to block foreign-antigen stimulated NFAT, NFKB and JNK.Inhibition of Al by FK506 can alone be sufficient to explain theanti-mitogenic effects of FK506, since activation in the presence ofFK506 is associated with increased B cell death.

[0295] The small number of foreign-response genes that are stilltriggered in the tolerance response can be inhibited or subverted frompro-mitogenic roles by other gene products in the tolerant cells. TheEgr-1 and Egr-2 transcription factors have been specifically implicatedin B cell mitogenesis, but are induced at lower levels in tolerant cellsthan after activation (FIG. 2B), a quantitative difference also true forEgr-1 protein. Their mitogenic activity in tolerant cells is likely tobe repressed by relatively high expression of NAB2, an Egr familyinhibitor, and it Egr/NAB2 heterodimers can activate tolerance-specificgenes. Inhibition by FK506 of Egr-2, and other sharedactivation/tolerance response genes such as CD72, shows that thisimmunosuppressive drug can also interfere with components of the activeself-tolerance response. This effect can further limit its efficacy inestablishing or restoring tolerance in autoimmunity and transplantation.

[0296] Many of the genes associated with the tolerance response can bepredicted to have negative regulatory functions for maintaining thetolerant state. The function of these genes in this context is unknownbut clues can be found in previous work. The largest change is anincrease in mRNA from Aeg-2 (also called CRISP-3), a gene known to becontrolled by Oct-2 in B cells which encodes a secreted protein ofunknown function. Two others, neurogranin and pcp-4, encode related geneproducts that have been implicated in regulation of calcium signalingthrough calmodulin and they can have a role in regulation of thedownstream effects caused by low level calcium spiking in tolerant cells(FIG. 4B). Two cell surface proteins upregulated in tolerant cellsregulate proximal signaling pathways necessary for the maintenance oftolerance. IgD is the primary receptor isotype expressed on tolerant Bcells, through which repeated binding of self antigen can triggercalcium oscillations. Proximal signaling can be decreased relative tonaive cells by increased levels of CD72, which has been shown to recruitthe inhibitory tyrosine phosphatase, SHP-1, and diminish BCR signaling(FIG. 4B). Increased IgD and CD72 in tolerant cells have been confirmedat the protein level.

[0297] The molecular definition of lymphocyte activation, tolerance, andFK506-immunosuppression established here can provide a guide to searchfor more efficient immunosuppressive drugs. In particular, the uniquetranscript signature associated with self-reactive cells can be used asa surrogate marker for tolerance, the phenotypes of which are not easilyassayed in a high throughput way. Recent advances in high throughputscreening techniques allow monitoring of gene expression after treatmentof cells with a chemical library of potential drug leads. By defining amolecular signature for peripheral tolerance, screens for new drugs thatbetter mimic the tolerance phenotype can be screened. With thisapproach, the drug target need not be known and need not be representedin the original expression screening platform, nor does the level oftranscript for the target protein itself need to change. To developdrugs that better emulate the active process of peripheral tolerance,the desired small molecule would suppress members of the activation-onlyearly response gene subset defined here, while leaving unaffected thesubset of early response genes that also participate in tolerance. Adrug with this profile could likely to block immunity but not tolerance,which can be key to (re)establishing immunological unresponsiveness inautoimmunity, allergy, or tranplantation.

REFERENCES

[0298] 1. Goodnow, C. C. et al. Self-tolerance checkpoints in Blymphocyte development. Adv Immunol 59, 279-368 (1995).

[0299] 2. Liu, J. et aL Calcineurin is a common target ofcyclophilincyclosporin A and FKBP-FK506 complexes. Cell 66, 807-15(1991).

[0300] 3. Kino, T. et al. FK-506, a novel immunosuppressant isolatedfrom a Streptomyces. I. Fermentation, isolation, and physico-chemicaland biological characteristics. J Antibiot (Tokyo) 40, 1249-55 (1987).

[0301] 4. Borel, J. F., Feurer, C., Gubler, H. U. & Stahelin, H.Biological effects of cyclosporin A: a new antilymphocytic agent. AgentsActions 6, 468-75 (1976).

[0302] 5. Wicker, L. S. et al. Suppression of B cell activation bycyclosporin A, FK506 and rapamycin. Eur J Immunol 20, 2277-83 (1990).

[0303] 6. Cooke, M. P. et aL Immunoglobulin signal transduction guidesthe specificity of B cell-T cell interactions and is blocked in tolerantself-reactive B cells. J Exp Med 179, 425-38 (1994).

[0304] 7. Healy, J. 1. et al. Different nuclear signals are activated bythe B cell receptor during positive versus negative signaling. Immunity6, 419-28 (1997).

[0305] 8. Rathmell, J. C., Fournier, S., Weintraub, B. C., Allison, J.P. & Goodnow, C. C. Repression of B7.2 on self-reactive B cells isessential to prevent proliferation and allow Fas-mediated deletion byCD4(+) T cells. J Exp Med 188, 651-9 (1998).

[0306] 9. Lipshutz, R. J., Fodor, S. P., Gingeras, T. R. & Lockhart, D.J. High density synthetic oligonucleotide arrays. Nat Genet 21, 20-4(1999).

[0307] 10. Seyfert, V. L., Sukhatme, V. P. & Monroe, J. G. Differentialexpression of a zinc finger-encoding gene in response to positive versusnegative signaling through receptor immunoglobulin in murine Blymphocytes. Mol Cell Biol 9, 2083-8 (1989).

[0308] 11. Newton, J. S. et al. B cell early response gene expressioncoupled to B cell receptor, CD40 and interleukin-4 receptorco-stimulation: evidence for a role of the Egr-2/krox 20 transcriptionfactor in B cell proliferation. Eur J Immunol 26, 811-6 (1996).

[0309] 12. Monroe, J. G. Up-regulation of c-fos expression is acomponent of the mlg signal transduction mechanism but is not indicativeof competence for proliferation. J Immunol 140, 1454-60 (1988).

[0310] 13. Huo, L. & Rothstein, T. L. Receptor-specific induction ofindividual AP-1 components in B lymphocytes. J Immunol 154, 3300-9(1995).

[0311] 14. Grumont, R. J., Rasko, J. E., Strasser, A. & Gerondakis, S.Activation of the mitogen-activated protein kinase pathway inducestranscription of the PAC-1 phosphatase gene. Mol Cell Biol 16, 2913-21(1996).

[0312] 15. Mittelstadt, P. R. & DeFranco, A. L. Induction of earlyresponse genes by cross-linking membrane Ig on B lymphocytes. J Immunol150, 4822-32 (1993).

[0313] 16. Hong, J. X., Wilson, G. L., Fox, C. H. & Kehrl, J. H.Isolation and characterization of a novel B cell activation gene. JImmunol 150, 3895-904 (1993).

[0314] 17. Mittrucker, H. W. et al. Requirement for the transcriptionfactor LSIRF/IRF4 for mature B and T lymphocyte function. Science 275,540-3 (1997).

[0315] 18. Grumont, R. J., Rourke, I. J. & Gerondakis, S. Rel-dependentinduction of A1 transcription is required to protect B cells fromantigen receptor ligationinduced apoptosis. Genes Dev 13, 400-11 (1999).

[0316] 19. Kuo, C. T., Veselits, M. L. & Leiden, J. M. LKLF: Atranscriptional regulator of single-positive T cell quiescence andsurvival. Science 277, 1986-90 (1997).

[0317] 20. Solvason, N. et al. Induction of cell cycle regulatoryproteins in anti-immunoglobulin-stimulated mature B lymphocytes. J ExpMed 184, 407-17 (1996).

[0318] 21. Marton, M. J. et al. Drug target validation andidentification of secondary drug target effects using DNA microarrays.Nat Med 4, 1293-301 (1998).

[0319] 22. Dolmetsch, R. E., Lewis, R. S., Goodnow, C. C. & Healy, J. I.Differential activation of transcription factors induced by Ca2+response amplitude and duration. Nature 386, 855-8 (1997).

[0320] 23. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T. &Saltiel, A. R. PD 098059 is a specific inhibitor of the activation ofmitogen-activated protein kinase in vitro and in vivo. J Biol Chem 270,27489-94 (1995).

[0321] 24. lida, S. et al. Deregulation of MUM1/IRF4 by chromosomaltranslocation in multiple myeloma. Nat Genet 17, 226-30 (1997).

[0322] 25. Langdon, W. Y., Harris, A. W., Cory, S. & Adams, J. M. Thec-myc oncogene perturbs B lymphocyte development in E-mu-myc transgenicmice. Cell 47,11-8 (1986).

[0323] 26. Svaren, J. et al. NAB2, a corepressor of NGFI-A (Egr-1) andKrox20, is induced by proliferative and differentiative stimuli. MolCell Biol 16, 3545-53 (1996).

[0324] 27. Pfisterer, P. et al. CRISP-3, a protein with homology toplant defense proteins, is expressed in mouse B cells under the controlof Oct2. Mol Cell Biol 16, 6160-8 (1996).

[0325] 28. Slemmon, J. R. et al. Camstatins are peptide antagonists ofcalmodulin based upon a conserved structural motif in PEP-19,neurogranin, and neuromodulin. J Biol Chem 271, 15911-7 (1996).

[0326] 29. Healy, J. I., Dohnetsch, R. E., Lewis, R. S. & Goodnow, C. C.Quantitative and qualitative control of antigen receptor signaling intolerant B lymphocytes. Novartis Found Symp 215, 137-44 (1998).

[0327] 30. Adachi, T., Flaswinkel, H., Yakura, H., Reth, M. & Tsubata,T. The B cell surface protein CD72 recruits the tyrosine phosphataseSHP-1 upon tyrosine phosphorylation. J Immunol 160, 4662-5 (1998).

[0328] 31. Tyagi, S. & Kramer, F. R. Molecular beacons: probes thatfluoresce upon hybridization. Nat Biotechnol 14, 303-8 (1996).

[0329] 32. Mason, D. Y., Jones, M. & Goodnow, C. C. Development andfollicular localization of tolerant B lymphocytes inlysozyme/anti-lysozyme IgM/IgD transgenic mice. Int Immunol 4, 163-75(1992).

[0330] Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications can be practiced within the scope of the appended claims.All publications and patent documents cited above are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

What is claimed is:
 1. A method of screening drug candidates comprising:a) providing a cell that expresses an expression profile gene selectedfrom the group selected of Egr-1, Egr-2, Nur77, c-myc, MIP-1a,MIP-1b,BL34, gfi-1, NAB2, neurogranin, SLAP, A1, E2-20K, SATB 1, Cctq,kappa V, pcp-4, TGIF, CD83, ApoE, Aeg-2, CD72, cyclin D2, 1ck, MEF-2C,brnk, IgD, Evi-2, vimentin, CD36, c-fes, c-fos, TRAP, hIP30, Ly6E.1,LRG-21, Fos B, gadd153, mafk, Ah-R, C/EBP beta, EZF, TIS7, TIS11,TIS11b, LSIRF, MKP1, PAC-1, PEP, MacMARCKS, SNK, Stra13, kir/gem, EB12,IL1-R2, MyD116, RP105, uPAR, 4F2, hRab30, Id3, BKLF, LKLF, EFP, bcl-3,caspase 2, GILZ, hIFI-204, hRhoH, TRAF5, LT-beta, IFNg-RII, gadd45,CDC47, NAG, scd2, kappa 0 ig, iap38, G7e, B29, and SCD2; b) adding adrug candidate to the cell; and c) determining the effect of the drugcandidate on the expression of the expression profile gene.
 2. A methodaccording to claim 1 wherein the determining comprises comparing thelevel of expression in the absence of the drug candidate to the level ofexpression in the presence of the drug candidate.
 3. A method accordingto claim 1 wherein the cell expresses an expression profile gene set ofat least one expression profile gene, and the effect of the drugcandidate on the expression of the set is determined.
 4. A methodaccording to claim 3 wherein the set comprises a tolerance setcomprising carb anh II, IgD, CD72, SATB1, ApoE, CD83, cyclin D2, Cctq,MEF-2C, TGIF, Aeg-2, Egr-1, lek, Egr-2, E2-20K, pcp-4, kappa V,neurogranin, NAB2, gfi-1 hIP-30, TRAP, bmk, CD36, Evi-2, vimetin,Ly6E.1, and c-fes.
 5. A method according to claim 4 wherein theexpression of hIP-30, TRAP, bmk, CD36, Evi-2, and c-fes are decreasedand the expression of carb anh II, CD72, SATB1, ApoE, CD83, cyclin D2,Cctq, MEF-2C, TGIF, Aeg-2, Egr-1, 1ck, Egr-2, E2-20K, pcp-4, kappa V,neurogranin, NAB2, gfi-1 are increased as a result of the introductionof the drug candidate.
 6. A method according to claim 3 wherein the setcomprises a stimulation set comprising Egr-1, Egr-2, NAB2, mafK, LRG-21,c-fos, c-myc, Stra13, AhR, gadd153, C/EBP beta, TIS11b, TIS11, gfi-1,EZF, Nur77, LSIRF, SNK, PAC-1, kir/gem, MacMARCKS, PEP, MKP1, hRab30,MIP-1b, MIP-1a, EB12, BL34, IL1-R2, TIS7, MyD116, A1, uPAR, RP105,Evi-24F2, CD72, Id3, BKLF, LKLF, EFP, Stat1, bcl-3, hRhoH, TRAF5, SLAP,LT-beta, IFNg-RII, GILZ. Caspase 2, gadd45, CDC47, NAG, scd2, kappa 0ig, B29, iap38, G7e, and hIFI-204.
 7. A method according to claim 6wherein the expression of Id3, BKLF, LKLF, EFP, Statl, bcl-3, hRhoH,TRAF5, SLAP, LT-beta, IFNg-RII, GILZ. Caspase 2, gadd45, CDC47, NAG,scd2, kappa 0 ig, B29, iap38, G7e, and hIFI-204 are decreased and theexpression of Egr-1, Egr-2, NAB2, mafK, LRG-21, c-fos, c-myc, Stra13,AhR, gadd153, C/EBP beta, TIS I lb, TISl 1, gfi-1, EZF, Nur77, LSIRF,SNK, PAC-1, kir/gem, MacMARCKS, PEP, MKP1, hRab30, MIP-1b, MIP-1a, EB12,BL34, IL1-R2, TIS7, MyD116, A1, uPAR, RP105, Evi-24F2, CD72 areincreased as aresult of the introduction of the drug candidate.
 8. Amethod according to claim 3 wherein the set comprises animmunosuppression set comprising hIFI-204, hRhoH, caspase 2, B29, SLAP,NAG, iap38, gadd45, BKLF, G7e, Id3, scd2, GILZ, Statl, kappa 0 ig,LT-beta, LKLF, IFNg-RII, mCDC47, EFP, TRAF5, and bcl-3.
 9. A methodaccording to claim 8 wherein the expression of hIFI-204, hRhoH, caspase2, B29, SLAP, NAG, iap38, gadd45, BKLF, G7e, Id3, scd2, GILZ, Stat1,kappa 0 ig, LT-beta, LKLF, IFNg-RII, mCDC47, EFP, TRAF5, and bcl-3 aredecreased and the expression of LSIRF, kir/gem, MKP1, hRab30, AhR,c-myc, Il1-R2, TIS11b,Evi2, A1, EB12, MyD116, MacMARCKS, MIP-1b, MIP-1a,PEP, CD72 are increased as a result of the introduction of the drugcandidate.
 10. A method according to claim 8 wherein theimmunosuppressive set further comprises c-fos, gadd153, EZF, C/EBP beta,Stra13, NAB2, mafK, and LRG-21.
 11. A method according to claim 10wherein the expression of c-fos, gadd153, EZF, C/EBP beta, Stra13, NAB2,mafK, and LRG-21 are increased as a result of the introduction of thedrug candidate.
 12. A method of screening for a bioactive agent capableof binding to a B lymphocyte modulator protein (BLMP), the methodcomprising combining the BLMP and a candidate bioactive agent, anddetermining the binding of the candidate agent to the BLMP.
 13. A methodaccording to claim 11 wherein the BLMP is selected from the groupconsisting of Egr-1, Egr-2, Nur77, c-myc, MIP-1a, MIP-1b,BL34, gfi-1,NAB2, neurogranin, SLAP, A1, E2-20K, SATBI, Cctq, kappa V, pcp-4, TGIF,CD83, ApoE, Aeg-2, CD72, cyclin D2, ick, MEF-2C, bmk, IgD, Evi-2,vimentin, CD36, c-fes, c-fos, TRAP, hIP30, Ly6E.1, LRG-21, Fos B,gadd153, mafK, Ah-R, C/EBP beta, EZF, TIS7, TIS 11, TIS11b, LSIRF, MKP1,PAC-1, PEP, MacMARCKS, SNK, Stra13, kir/gem, EB12, IL1-R2, MyD116,RP105, uPAR, 4F2, hRab30, Id3, BKLF, LKLF, EFP, bcl-3, caspase 2, GILZ,hIFI-204, hRhoH, TRAF5, LT-beta, IFNg-RII, gadd45, CDC47, NAG, scd2,kappa 0 ig, iap38, G7e, B29, and SCD2.
 14. A method for screening for abioactive agent capable of modulating the activity of a B lymphocytemodulator protein (BLMP), the method comprising combining the BLMP and acandidate bioactive agent, and determining the effect of the candidateagent on the bioactivity of the BLMP.
 15. A method according to claim 13wherein the BLMP is selected from the group consisting of Egr-1, Egr-2,Nur77, c-myc, MIP-1a, MIP-1b,BL34, gfi-1, NAB2, neurogranin, SLAP, A1,E2-20K, SATBI, Cctq, kappa V, pcp-4, TGIF, CD83, ApoE, Aeg-2, CD72,cyclin D2, 1ck, MEF-2C, bmk, IgD, Evi-2, vimentin, CD36, c-fes, c-fos,TRAP, hIP30, Ly6E.1, LRG-21, Fos B, gadd153, mafK, Ah-R, C/EBP beta,EZF, TIS7, TIS11, TIS11b, LSIRF, MKPl, PAC-1, PEP, MacMARCKS, SNK,Stra13, kir/gem, EB12, IL1-R2, MyD116, RP105, uPAR, 4F2, hRab30, Id3,BKLF, LKLF, EFP, bcl-3, caspase 2, GILZ, hIFI-204, hRhoH, TRAF5,LT-beta, IFNg-RII, gadd45, CDC47, NAG, scd2, kappa 0 ig, iap38, G7e,B29, and SCD2.
 16. A method of evaluating the effect of animmunosuppressive drug comprising: a) administering the drug to apatient; b) removing a cell sample from the patient; and c) determiningthe expression profile of the cell sample.
 17. A method according toclaim 16 further comprising comparing the expression profile to anexpression profile of a healthy individual.
 18. A method according toclaim 16 wherein the expression profile includes at least one geneselected from the group consisting of Egr-1, Egr-2, Nur77, c-myc,MIP-1a, MIP-1b,BL34, gfi-1, NAB2, neurogranin, SLAP, A1, E2-20K, SATB1,Cctq, kappa V, pcp-4, TGIF, CD83, ApoE, Aeg-2, CD72, cyclin D2, 1ck,MEF-2C, bmk, IgD, Evi-2, vimentin, CD36, c-fes, c-fos, TRAP, hIP30,Ly6E.1, LRG-21, Fos B, gadd153, mafK, Ah-R, C/EBP beta, EZF, TIS7,TIS11, TIS11b, LSIRF, MKP1, PAC-1, PEP, MacMARCKS, SNK, Stra13, kir/gem,EB12, IL1-R2, MyD116, RP105, uPAR, 4F2, hRab30, Id3, BKLF, LKLF, EFP,bcl-3, caspase 2, GILZ, hIFI-204, hRhoH, TRAF5, LT-beta, IFNg-RII,gadd45, CDC47, NAG, scd2, kappa 0 ig, iap38, G7e, B29, and SCD2.
 19. Anarray of probes, comprising a support bearing a plurality of nucleicacid probes complementary to a plurality of mRNAs fewer than 1000 innumber, wherein the plurality of mRNA probes includes an mRNA expressedby a gene selected from the group consisting of Egr-1, Egr-2, Nur77,c-myc, MIP-1a, MIP-1b, BL34, gfi-1, NAB2, neurogranin, SLAP, A1, E2-20K,SATB1, Cctq, kappa V, pcp-4, TGIF, CD83, ApoE, Aeg-2, CD72, cyclin D2,1ck, MEF-2C, bmk, IgD, Evi-2, vimentin, CD36, c-fes, c-fos, TRAP, hIP30,Ly6E.1, LRG-21, Fos B, gadd153, mafK, Ah-R, C/EBP beta, EZF, TIS7,TIS11, TIS11b, LSIRF, MKP1, PAC-1, PEP, MacMARCKS, SNK, Stra13, kir/gem,EB12, IL1-R2, MyD116, RP105, uPAR, 4F2, hRab30, Id3, BKLF, LKLF, EFP,bcl-3, caspase 2, GILZ, hIFI-204, hRhoH, TRAF5, LT-beta, IFNg-RII,gadd45, CDC47, NAG, scd2, kappa 0 ig, iap38, G7e, B29, and SCD2.
 20. Thearray of claim 19 , wherein the probes are cDNA sequences.
 21. The arrayof claim 19 , comprising a plurality of sets of probes, each set ofprobes complementary to subsequences from a mRNA.